TOWARDS IDENTIFYING GENES INVOLVED IN PHOTOSYNTHETIC ACCLIMATION TO LOW
TEMPERATURES
By
Gary Rudd Larson

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Genetics – Master of Science
2014

ABSTRACT
TOWARDS IDENTIFYING GENES INVOLVED IN PHOTOSYNTHETIC ACCLIMATION TO LOW
TEMPERATURES
By
Gary Rudd Larson
This thesis describes the use of the Dynamic Environmental Phenotype Imaging (DEPI) system – an
imaging system that allows for dynamic environmental control and continuous monitoring of multiple
photosynthetic parameters, including photosynthetic efficiency (II) and non-photochemical quenching
(NPQ), to probe the responses and acclimation of photosynthesis in Arabidopsis thaliana (hereafter
referred to as Arabidopsis) to low temperature. Acute (minutes to hours) exposure to low, non-freezing
temperatures dramatically decreased II and increased NPQ. Under these conditions, qE – the energydependent quenching mechanism – decreased with increasing light, indicating a breakdown of the normal
regulation of the light reactions. The photosynthetic processes recovered progressively during acclimation
over a period of weeks. The effects of cold and subsequent acclimation responses were highly
heterogeneous. Also, there was natural variation in photosynthetic acclimation among five Arabidopsis
accessions tested. Altered photosynthetic acclimation to low temperatures was also observed in transgenic
Arabidopsis lines overexpressing three transcription factors – HSFC1, CRF3, and AS1 – and in
Arabidopsis mutant lines carrying knock out mutations of the CAMTA1, CAMTA2, and CAMTA3
transcription factors. Taken together, the results presented in this thesis provide strong evidence that the
DEPI technology can be used in combination with Arabidopsis genetic variation to identify genes that
condition the fundamental process of photosynthetic acclimation to low temperatures.

This work is dedicated to my wife, Elise, and my three children, Remi, Max, and Brynn, without whom
this would not have been possible

iii

ACKNOWLEDGMENTS

I would like to extend special thanks to Dr. Mike Thomashow and Dr. Dave Kramer for all the help and
guidance they gave me. I would also like to thank the rest my thesis committee, Dr. Christoph Benning,
Dr. Sheng Yang He, and Dr. Tom Sharkey, for their time and help. I’d like to thank the Schemske Lab for
their help it the Sweden and Italy ecotypes and RIL lines. I’d like to thank the engineers in the Kramer
Lab as well for their help with DEPI chambers anytime there were any problems or I needed it to do
something new.

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TABLE OF CONTENTS

LIST OF FIGURES

vii

CHAPTER 1: PHOTOSYNTHETIC ACCLIMATION TO LOW TEMPERATURES
Introduction
Results
Chlorophyll Fluorescence Parameters Rapidly Change in Response to Temperature
Chlorophyll Fluorescence Parameters Acclimate after Extended Exposure to Low
Temperatures
Discussion
Materials and Methods
Plants
Fluorescence Measurements
REFERENCES
CHAPTER 2: NATURAL VARIATION OF PHOTSYNTHETIC ACCLIMATION TO LOW
TEMPERATURES
Introduction
Results
Natural Variation in Photosynthetic Parameters Among Five Accessions
of Arabidopsis
Natural Variation in the Rapid Response of Chlorophyll Fluorescence
Parameters to Low Temperatures
Natural Variation in the Acclimation of Photosynthesis to Low Temperatures
There is little Natural Variation in Fluorescence Parameters after Cold Treatment
FV/FM is Highly Variable between Accessions of Arabidopsis at Low Temperature
Discussion
Materials and Methods
Plant Materials
Fluorescence Measurements
REFERENCES
CHAPTER 3: MUTANT SCREENS FOR VARIATIONS IN PHOTOSYNTHETIC
ACCLIMATION TO LOW TEMPERATURES
Introduction
Results
Identification of Transcription Factors that Affect Chlorophyll Fluorescence
Parameters at Warm Temperatures and in the Cold
Differences in Photosynthetic Properties in Transcription Factor Mutants and
Overexpression Lines at 22oC and at 4oC
CAMTA Mutants Show Differences in Fluorescence Parameters at Warm
Temperatures and in the Cold
Discussion
Materials and Methods
Plant Materials
Fluorescence Measurements
REFERENCES
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CHAPTER 4: OVERALL CONCLUSIONS

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LIST OF FIGURES

Figure 1 Changes in Photosynthetic Parameters in Response to Low Temperature

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Figure 2 Changes in Photosynthetic Efficiency During a Five Week Exposure
to Low Temperature

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Figure 3 Changes in Non-photochemical Quenching During a Five Week Exposure
to Low Temperature

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Figure 4 Changes in the Relationship between qE and Light Intensity in Response
to Low Temperature

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Figure 5 Photosynthetic Acclimation to Low Temperature Varies with Leaf
Developmental Stage

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Figure 6 Fluorescence Parameters of Five Arabidopsis Accessions

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Figure 7 Immediate Response of Fluorescence Parameters of Five Arabidopsis
Accessions to a Decrease in Temperature

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Figure 8 Acclimation Response of Fluorescence Parameters of Five Arabidopsis
Accessions after Three Weeks at 4oC

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Figure 9 Post-cold Fluorescence Parameters of Five Arabidopsis Accessions

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Figure 10 Acclimation of FV/FM to Low Temperature in Five Accessions of Arabidopsis

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Figure 11 II in Cold Acclimation Associated Transcription Factors at Three Time Points

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Figure 12 NPQ in Cold Acclimation Associated Transcription Factors at Three Time Points

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Figure 13 qE and qI in Cold Acclimation Associated Transcription Factors
at Three Time Points

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Figure 14 Photosynthetic Parameters in Seven Arabidopsis Mutants at Three Time Points

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Figure 15 Photosynthetic Parameters in CAMTA Mutants under Consistent Light
at Four Time Points

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Figure 16 Photosynthetic Parameters in CAMTA Mutants under Sinusoidal Light
at Four Time Points

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Figure 17 Photosynthetic Parameters in CAMTA Mutants under Fluctuating Light
at Four Time Points

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Figure 18 Fluctuating Day Light Intensities for CAMTA Experiment

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viii

CHAPTER 1: PHOTOSYNTHETIC ACCLIMATION TO LOW TEMPERATURES
Introduction
Photosynthesis is essential to power most life in our ecosystem. It is also a delicate process that can
damage the organisms that rely on it. They must balance photochemistry, those processes involving the
absorption of light energy and converting it to chemical energy, with the use of energy for carbon
fixation. When these processes are out of balance, dangerous products like reactive oxygen species are
generated. Photosynthetic balance is often disrupted when plants are exposed to stressful conditions. A
common stress for many agriculturally important plants is low temperature.

Low temperature poses a major problem for plant photosynthetic balance. As temperatures decrease,
carbon assimilation by the Calvin-Benson Cycle is diminished (Hurry et al., 1994; Huner et al., 1993).
The decrease in carbon fixation can be partially explained by a decrease in enzymatic rates of CalvinBenson Cycle enzymes (Strand et al., 1997). To maintain balance and prevent photodamage,
photochemistry must also decrease at low temperatures. These changes in photosynthesis can be probed
by measuring the amount of oxygen generated through water splitting by the oxygen evolving complex
(OEC), or the yield of chlorophyll fluorescence, measuring the fraction of absorbed light energy reemitted as fluorescence from excited chlorophylls rather than being used in photochemistry or radiated as
heat (Strand et al., 1997; Greer et al., 1991; Huner et al., 1993). Oxygen evolution levels decrease at low
temperatures (Öquist et al., 1993; Huner et al., 1993).

The operating efficiency of photosystem II (PSII), an estimate of the efficiency of light absorbed by PSII
being used for photochemistry (II), tends to be lower in plants exposed to low temperature compared
with warm temperature controls (Strand et al., 1997; Hurry et al., 1994). This decreased yield is typically
accompanied by higher extents of non-photochemical quenching (NPQ), the processes used to dissipate
excess energy absorbed by light harvesting complexes (LHCs) not used for photochemistry (Hurry et al.,

1

1994; Adams et al., 1994). In higher plants, NPQ is composed of two main parameters: energy-dependent
quenching (qE) and photoinhibition (qI). qE involves the energy-driven acidification of the thylakoid
lumen, which triggers the conversion of the violaxanthin to zeaxanthin and the protonation of PsbS to
dissipate energy from the excited chlorophylls in the LHCs (Müller et al., 2001). This process is rapidly
(within a few minutes) reversible in the dark as proton efflux decreases the acidity of the thylakoid lumen
decreases. The qI quenching is associated with damage and repair of PSII and recovers only slowly (over
the hours time scale) when high light stress is removed from plants. Maximum PSII efficiency (FV/FM),
the maximal efficiency at which light absorbed by PSII can be used for photochemistry, is also lower
when temperatures decrease (Adams et al., 1994; Hurry et al., 1992; Greer et al., 1991). This is indicative
of either unrepaired damage to PSII due to excess light energy not being dissipated safely by NPQ, or a
lack of reversal of qE from the previous day. This persistent qE might prepare the plant for next day of
light stress to better protect the plant early in the day (Adams et al., 1994).

With prolonged exposure to low temperatures (days to weeks), some plants are able to increase
photosynthetic rates. This process is known as photosynthetic acclimation to low temperature.
Photosynthetic acclimation to low temperature is characterized by increased sucrose synthesis, increased
carbon fixation, and increased photochemistry. One way this is accomplished is by increasing amounts of
enzymes involved in carbon assimilation and sucrose synthesis to compensate for decreased enzymatic
rates (Strand et al., 1997; Hurry et al., 1994). Plants acclimated to low temperature for 10 days show an
increase in sucrose-phosphate synthase and cytosolic fructose-1,6-bisphosphotase, two enzymes in the
sucrose synthesis pathway (Hurry et al., 1994; Strand et al., 1999). They also have elevated amounts of
Rubisco and several other enzymes in the Calvin-Benson Cycle (Strand et al., 1999). This increases
carbon fixation rates, which also increases the amount of absorbed light needed for photochemistry.
FV/FM and II also increase after prolonged exposure to low temperatures, while NPQ decreases (Strand et
al., 1997; Hurry et al., 1994, Greer et al., 1991). Leaves fully developed at low temperatures, rather than

2

being transferred to low temperatures after development at warm temperatures, have even higher rates of
photosynthesis which are higher than those of control plants at warm temperatures (Strand et al., 1997).
Also, leaves adapted to low temperature show increased rates of photosynthesis – measured as carbon
assimilation, oxygen evolution, or chlorophyll fluorescence – when tested at warm temperatures,
compared to control plants constantly kept at warm temperatures (Strand et al., 1997).

In order to measure the rapid response of photosynthesis to a decrease in temperature, I needed the ability
to probe photosynthetic parameters with sufficient resolution to observe responses to rapid changes in
environmental conditions. The Dynamic Environmental Phenomic Imaging (DEPI) system is being
developed by the Kramer Lab to make high throughput phenomic measurements under a variety of
environmental conditions similar to those experienced in the field, rather than under the normal lab
conditions. The DEPI system is set up to make chlorophyll fluorescence images at multiple light
intensities and variable temperatures. I used the DEPI system to measure fluorescence responses to
changes in temperature and light intensity for short-term and long-term experiments using Arabidopsis
thaliana (hereafter referred to as Arabidopsis) as a model test organism. I was able to measure II, NPQ,
qE, and qI continuously throughout the duration of these experiments, without disturbing the plants other
than changing the ambient temperature in the DEPI chamber.

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Results
Chlorophyll Fluorescence Parameters Rapidly Change in Response to Temperature
Using the newly developed DEPI system I was able to measure chlorophyll fluorescence responses that
reflect photosynthesis to changes in temperature. The rapid response of Arabidopsis Col-0 to a decrease in
temperature from 22oC to 4oC was measured monitoring II, NPQ, qE, and qI. As the temperature
decreased, II decreased and NPQ increased (Figure 1). The two major components of NPQ, qE and qI,
increased as well. While the decrease in PSII operating efficiency appeared to level out when the
temperature did, NPQ continued to increase throughout the measurement time.

Chlorophyll Fluorescence Parameters Acclimate after Extended Exposure to Low Temperatures
Using the DEPI system, I also measured acclimation to low temperature in II, NPQ, qE, and qI in
Arabidopsis Col-0 plants during a five week exposure to 4oC. The DEPI system allowed me to measure
all four of these parameters continuously over the length of the experiment, without the need to disturb
the plants. FV/FM and II decreased in Col-0 plants in response to a drop in temperature from 22oC to 4oC
(Figure 2). After several days, plants began showing signs of acclimation, continuing through five weeks
at low temperature. When plants were returned to 22oC, they showed higher rates of II than before the
acclimation. The value of II reached its minimum almost immediately after the change in temperature,
but FV/FM continued to decrease for over a week. II began to slowly increase after approximately 3 days
exposure to cold, recovering to about 60% of warm temperature values after five weeks at 4oC. FV/FM did
not begin to recover until almost 14 days at low temperature, but it recovered to approximately 90% of
22oC values by week five. After returning plants to 22oC, both II and FV/FM showed an increase over precold treatment values.

4

Figure 1 Changes in Photosynthetic Parameters in Response to Low Temperature
The rapid response of Arabidopsis Col-0 to a decrease in temperature from 22oC to 4oC.
The temperature was set to 4oC at 15:00 (black arrows), and took approximately 30
minutes to level out around 4oC (blue arrows). II decreased immediately as temperature
decreased (A), while NPQ and its components, qE and qI, increased (B, C, D
respectively). Only two measurements were made for qE and qI to reduce the time the
lights were off. n=4

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Figure 2 Changes in Photosynthetic Efficiency During a Five Week Exposure to Low Temperature
FV/FM (A) and II at about 120 mol photons m-2 s-1 (B) of Arabidopsis Col-0 during five
weeks at 4oC. n=2
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When exposed to a drop in temperature, NPQ increased rapidly (Figure 3). This increase in NPQ was
concurrent with increases in both the qE and qI components. NPQ decreased again by day 2 at low
temperature, but recovered back to nearly five-fold the 22oC value by day 4 (Figure 3). The qE component
also quickly recovered at low temperature, but the qI component remained high for several weeks (Figure
3). When plants were returned to 22oC, NPQ, qE, and qI were all negligible.

When plants were exposed to a decrease in temperatures, qE decreased as light intensity increased (Figure
4). This was altered when compared to pre-cold treatment, where qE increased as light intensity increased.
After three weeks at 4oC, this relationship began to recover, though it was still altered at mid-high-light
intensities. When plants were returned to 22oC, the relationship was restored, but qE increased to much
lower values at high light intensity.

When plants were exposed to a drop in temperature, all leaves decreased in PSII efficiency, but older
leaves appeared to be more affected (Figure 5). As plants acclimated, younger leaves, especially those
developed at low temperatures, recovered more quickly than did older leaves. When plants were returned
to 22oC, all leaves appeared to have higher photosynthesis than the leaves before cold-treatment.

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Figure 3 Changes in Non-photochemical Quenching During a Five Week Exposure to Low
Temperature
NPQ (A), qE (B), and qI (C) of Arabidopsis Col-0 at about 120 mol photons m-2 s-1
during five weeks at 4oC. n=2

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Figure 4 Changes in the Relationship Between qE and Light Intensity in Response to Low
Temperature
The relationship between qE and light intensity in Arabidopsis Col-0 before cold
treatment (A), immediately after cold (B), after three weeks at 4oC (C), and after
returning to 22oC (D). n=2

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Figure 5 Photosynthetic Acclimation to Low Temperature Varies with Leaf Developmental Stage
False color II images of an Arabidopsis Col-0 plant at approximately 120 mol photons
m-2 s-1 before cold (A), immediately after cold (B), after three weeks at 4oC (C), and after
returning to 22oC (D).
10

Discussion
When Arabidopsis plants experienced a decrease in temperature, I observed rapid changes in
photosynthetic parameters as measured by chlorophyll a fluorescence, with rapid decreases in II and
increases in both rapidly (qE) and slowly (qI) relaxing forms of NPQ. The increased NPQ presumably
serves to protect the plants from increased excess light energy resulting from decreased photosynthetic
capacity at low temperatures. A reasonable possible explanation for the observed increased qE is a buildup
of protons in the lumen, resulting from decreased ATP synthase activity. Preliminary results (not shown)
showing strong decreases in ATP synthase activity in vivo are consistent with this interpretation
(Kanazawa et al., 2002). I hypothesize that the ATP synthase itself could be temperature sensitive.
Alternatively, the rates of carbon fixation decreased, and a negative feedback mechanism slowed down
the turnover of the ATP synthase.

Over a six week experiment, I probed acclimation o f photosynthesis to low temperature by measuring
changes in chlorophyll fluorescence to prolonged exposure to low temperatures. The temperature was
lowered to 4oC and plants were kept at this temperature for approximately five weeks. Photosynthetic
efficiency dropped immediately upon the change in temperature, but after about five days it began to
recover. NPQ increased immediately when the change in temperature occurred, presumably to dissipate
the excess light energy due to the decrease in photosynthetic efficiency. II acclimated slowly over the
entire five week cold treatment, while a majority of NPQ recovered rapidly (within one week). This
apparent decrease in NPQ without an increase in the efficiency of absorbed energy being used in
photosynthesis could be caused by an increase in damaged PSII. Damaged PSII centers would absorb
light which would neither be used for photochemistry nor dissipated through NPQ. This is supported by
the fact that FV/FM continued to decrease during this time, which could have resulted from an increase in
damaged PSII. The acclimation of II to low temperatures could have been due to an increase in carbon
metabolism proteins previously reported (Strand et al., 1999). As carbon metabolism protein activity

11

increases, the demand for dissipation of energy by NPQ would decrease and the demand for the increased
flow of absorbed energy through photochemistry would also increase.

Acclimation of II could also be caused by a decrease in the amount of energy absorbed by PSII. If the
absolute amount of photochemistry being performed by the plant remained the same, this decrease in
absorbed energy would result in an apparent increase in II, because the fraction of absorbed light being
used for photochemistry would increase. The increased levels of anthocyanin reported at low temperature
could contribute to this because anthocyanins can absorb the light that would alternatively be absorbed by
photosynthetic complexes (Huner et al., 1998). Lower energy absorption could also be caused by a
decrease in antenna complexes, which would limit the amount of light absorbed.

qE and qI, the two major components of NPQ in higher plants, also acclimated in response to low
temperatures. The response of qE to increases in light intensity was significantly altered at low
temperature. At 22oC, qE increased as light intensity increased. This is expected because as light intensity
increases, more electrons are passed through the electron transport chain of photosynthesis, and
consequently more protons are pumped into the thylakoid lumen. This decreases the pH, which triggers
the conversion of violaxanthin to zeaxanthin and protonates PsbS, the mechanisms of qE that dissipates
excess light energy. When temperatures were lowered to 4oC however, qE decreased with increased light
intensity. As qE acclimated, it began to increase in response to increased light intensities once again. This
change in the qE response to increased light intensity could have been due to a failure to decrease lumen
pH or a lack of proton motive force (pmf). pmf is the force generated by the buildup of protons against a
concentration gradient. In photosynthesis, this allows the ATP synthase to rotate as protons flow out with
the concentration gradient through the ATP synthase. Altered qE response to changes in light intensity
could also have been due to a change in the xanthophyll cycle itself. If the conversion of violaxanthin to

12

zeaxanthin and back is affected, this would affect qE levels or the ability of qE to respond to increases in
light intensity.

When plants were returned to 22oC, II was higher than before the cold treatment, while NPQ and its
components were lower. Changes made during acclimation to low temperature made the plants more
efficient photosynthetically when they were returned to higher temperatures. This could have resulted
from a reduction in antenna complex number or an increase in carbon metabolism that continued to occur
when plants were returned to warm temperatures.

Using the DEPI system I also saw that photosynthetic acclimation to low temperatures differed in leaves
at different developmental stages. Young leaves developed mostly or entirely at low temperature
recovered first at low temperature. Some old leaves were able to acclimate as well, though not to the same
levels as leaves developed after the transfer to low temperature. This indicates that mature leaves have a
limited ability to acclimate to low temperatures, but new leaves can be ‘programmed’ to perform better
under the low temperature conditions. All of the leaves acclimated to low temperatures showed higher II
when returned to 22oC than they did prior to low temperature treatment. This is supported by experiments
that compared leaves developed at low temperature with those acclimated to low temperature or at warm
temperature. Leaves developed at low temperature had the highest rates of photosynthesis in these
experiments (Strand et al., 1997). It’s possible this occurs because the acclimation mechanisms which
increase photosynthetic efficiency at low temperatures do not return to their original, pre-cold treatment
states.

The DEPI system can be used to measure photosynthetic acclimation to low temperature. This will enable
interested investigators to use the DEPI system to do genetic screens to identify important genes in the

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acclimation of photosynthesis to low temperatures and better understand the mechanisms of
photosynthetic acclimation to low temperature.

14

Materials and Methods
Plant Materials
Arabidopsis thaliana Col-0 were grown in soil at 22oC under a light intensity of around 120 mol photons
m-2 s-1 for eight hours each day. After three weeks plants were moved into the DEPI chamber for
measurement. To measure the short-term response, plants were monitored for several days at 22oC, and
then the temperature was dropped to 4oC at 15:00. The temperature change took about 30 minutes. For the
acclimation experiment, the plants were monitored for several days at 22oC, then the temperature was
changed to 4oC at 13:00. Plants stayed at 4oC for 38 days, and then temperatures were raised to 22oC at
13:00 for the duration of the experiment. Plants were at approximately 120 mol photons m-2 s-1 until
14:00, then went to 250 mol photons m-2 s-1 until 15:00, 500 mol photons m-2 s-1 until 16:00, and 750
mol photons m-2 s-1 until 17:00 for the qE response measurements.

Fluorescence Measurements
For the immediate response experiment, II images were taken every five minutes. qE measurments were
taken once before and once after the temperature change. For each qE/qI measurement, the lights were
turned off for ten minutes. For the acclimation experiment, II images were taken twice at 120 mol
photons m-2 s-1, and once at each increased light intensity. qE images were taken once at the end of each
light intensity period to measure qE response to changes in light intensity. Calculations were made using
the Visual Phenomic program according to established protocols (Baker et al., 2007; Genty et al., 1989).
II = (Fm’-Fs)/Fm’
NPQ = (Fm-Fm’)/Fm’
qE = (Fm’’-Fm’)/Fm’
qI = (Fm-Fm’’)/Fm’’

15

REFERENCES

16

REFERENCES

Adams III, W.W., Demmig-Adams, B., Verhoeven, A.S. and Barker, D.H. (1994) ‘Photoinhibition’
During Winter Stress: Involvement of Sustained Xanthophyll Cycle-dependent Energy Dissipation. Aust.
J. Plant Physiol. 22, 261-276.
Baker, N.R., Harbinson, J. and Kramer, D.M. (2007) Determining the limitations and regulation of
photosynthetic energy transduction in leaves. Plant, Cell and Environment, 30, 1107-1125.
Genty, B., Briantais, J.M. and Baker, N.R. (1989) The relationship between the quantum yield of
photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica
Acta. 990, 87-92.
Greer, D.H., Ottander, C. and Öquist, G. (1991) Photoinhibition and recovery of photosynthesis in intact
barley leaves at 5 and 20oC. Physiologia Plantarum, 81, 203-210.
Huner, N.P.A., Öquist, G., Hurry, V.M., Krol, M., Falk, S. and Griffith, M. (1993) Photosynthesis,
photoinhibition, and low temperature acclimation in cold tolerant plants. Photosynthesis Research, 37,
19-39.
Huner, N.P.A., Öquist, G. and Sarhan, F. (1998) Energy balance and acclimation to light and cold. Trends
in Plant Science, 3, 224-230.
Hurry, V.M., Krol, M., Öquist, G. and Huner, N.P.A. (1992) Effect of long-term photoinhibition on
growth and photosynthesis of cold-hardened spring and winter wheat. Planta, 188, 369-375.
Hurry, V.M., Malmberg, G., Gardestrom, P. and Öquist, G. (1994) Effects of a Short_Term Shift to Low
Temperature and of Long-Term Cold Hardening on Photosynthesis and Ribulose-1,5-Bisphosphate
Carboxylase/Oxygenase and Sucrose Phophate Synthase Activity in Leaves of Winter Rye (Secale
cereale L.). Plant Physiol. 106, 983-990.
Kanazawa, A. and Kramer, D.M. (2002) In vivo modulation of nonphotochemical exciton quenching
(NPQ) by regulation of the chloroplast ATP synthase. Proc Natl Acad Sci U S A. 99, 12789-12794.
Müller, P., Li, X.P. and Niyogi, K.K. (2001) Non-Photochemical Quenching. A Response to Excess Light
Energy. Plant Physiol. 125, 1558-1566.
Öquist, G., Hurry, V.M. and Huner, N.P.A. (1993) Low-Temperature Effects on Photosynthesis and
Correlation with Freezing Tolerance in Spring and Winter Cultivars of Wheat and Rye. Plant Physiol.
101, 245-250.
Strand, Å., Hurry, V., Gustafsson, P. and Gardestrom, P. (1997) Development of Arabidopsis thaliana
leaves at low temperatures releases the suppression of photosynthesis and photosynthetic gene expression
despite the accumulation of soluble carbohydrates. The Plant Journal, 12, 605-614.
Strand, Å., Hurry, V., Henkes, S., Huner, N., Gustafsson, P., Gardestrom, P. and Stitt, M. (1999)
Acclimation of Arabidopsis Leaves Developing at Low Temperatures. Increasing Cytoplasmic Volume

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Accompanies Increased Activities of Enzymes in the Calvin Cycle and in the Sucrose-Biosynthesis
Pathway. Plant Physiol. 119, 1387-1397.

18

CHAPTER 2: NATURAL VARIATION OF PHOTOSYNTHETIC ACCLIMATION TO LOW
TEMPERATURES
Introduction
A balance between photochemistry and the demands of carbon metabolism is necessary for plant growth
and development. Many biotic and abiotic stresses can disrupt balanced photosynthesis. One stress that
affects many plants, including commercially important crops, is low temperature. In order to survive and
develop under long periods of cold, some plants are able to increase photosynthetic rates while
maintaining the necessary balance in photosynthesis.

Understanding the genetic basis of photosynthetic acclimation to low temperature will provide greater
clarity on the mechanisms of this acclimation and may allow for its improvement in some plants.
To identify the genetic basis of a trait, there must be genetic variation. This variation can be natural
variation, meaning it exists in nature without human manipulation, or it can be introduced through
mutagenesis. Natural selection can act on genetic variation to allow adaptation to a local environment.
Photosynthesis is one characteristic that is highly variable in plant species, and even within a given
species. Natural variation in several photosynthetic parameters has been identified, including NPQ and
photosynthetic acclimation to low temperatures (Öquist et al., 1993; Alonso-Blanco et al., 2000; Flood et
al., 2011).

Natural variation can be exploited in several ways to identify the genes underlying many complex traits.
Genome-wide association studies (GWAS) uses large numbers of plant accessions to associate a
phenotype with a quantitative trait locus (QTL) (Atwell et al., 2010). Genome-wide association studies
require large numbers of genotyped lines (Atwell et al., 2010). These lines can then be screened for a
phenotype, and genetic markers can be associated with a phenotype. To demonstrate the potential of
GWAS, Atwell et al. surveyed 107 phenotypes in more than 95 Arabidopsis accessions. These
phenotypes included time of flowering under different conditions, defense against pathogens, mineral
19

concentrations, and development. The investigators were able to identify many QTL associated with these
traits. However, this method has some drawbacks. For instance, population structure can skew results and
give false associations (Atwell et al., 2010). False associations occur because closely related lines will
have high genetic similarity that is unrelated to the trait of interest. Care must be taken to avoid
population structure problems, or methods must be used to account for it (Atwell et al., 2010).

Recombinant inbred line (RIL) studies harness the power of recombination to isolate QTLs (Mauricio,
2001). RIL analysis requires two parental lines which demonstrate variation in the desired trait whose
genotypes are known. These lines are crossed and the next generation is selfed for about eight generations
to generate a mapping population (Mauricio, 2001). The mapping population can then be genotyped in
relation to the two parent lines and screened for the desired phenotype. RIL lines with the phenotype are
analyzed to identify genomic markers from the parents that are associated with the phenotype. Increased
marker density and more recombination events (more RIL lines) can greatly increase the precision of the
QTL detection in these experiments, up to a certain amount (Mauricio, 2001). In 1998, Beavis showed
that increasing the population size from 100 to 500 individuals had large results, but the gains from going
from 500 to 1000 individuals were relatively small (Beavis, 1998). RIL analysis is commonly used in
plant genetics. Experiments between naturally occurring ecotypes, accessions of a species adapted to their
local environment, of Arabidopsis thaliana from Sweden (SW) and Italy (IT) have been used to identify
QTL for flowering time (Grillo et al., 2013). In this experiment, the investigators found three QTLs
associated with flowering without a vernalization requirement, and six QTLs related to flowering with a
vernalization treatment. There was one QTL that overlapped between the vernalization and
nonvernalization QTLs.

Other mapping populations have also been used to identify QTLs in photosynthesis (Jung et al., 2009).
Sixty-two accessions of Arabidopsis were measured for NPQ, and classified as being high or low. A high
NPQ accession was crossed with a low NPQ accession and the second offspring generation (F2
20

generation) was screened. Two QTLs were found, one on chromosome 1 and one on chromosome 2. One
of the QTLs was verified by making near-isogenic lines containing the low NPQ background genotype
with only the QTL region of the high NPQ accession combined in. These lines showed high NPQ,
verifying the effect of this QTL on NPQ.

In the experiments described below, I used the DEPI system to screen five accessions of Arabidopsis –
collected from geographical locations that differed greatly in climate – to determine whether I could
identify natural variation in photosynthetic acclimation to low temperatures.

21

Results
Natural Variation in Photosynthetic Parameters Among Five Accessions of Arabidopsis
Prior to cold treatment, all five accessions of Arabidopsis tested – Col-0, IT, SW, WS-2, and Ler – had
high II values and low NPQ values (Figure 6). These accessions represent a range in the natural habitat
of Arabidopsis, from SW at the northern edge to IT at the southern. They also represent a range of
climates, with SW plants regularly experiencing temperatures below 0oC during the winter, while plants
in the IT habitat only experienced sub-freezing temperatures once in the eight years monitored (Oakley et
al., 2014). Increasing light intensity at the end of the day caused a decrease in efficiency and an increase
in all quenching parameters (Figure 6). Col-0 and Ler had the highest II at all light intensities and SW
had the lowest. At low light intensities IT was intermediate in II, but at high light intensities it decreased
to values similar to SW. The variation between accessions was larger at low light intensity. NPQ showed
little variation at low light intensity, but had more variability at higher light intensities. IT had the lowest
NPQ at all light intensities. There was little variation in qE and qI, but IT had the lowest qI at all light
intensities.

Natural Variation in the Rapid Response of Chlorophyll Fluorescence Parameters to Low
Temperatures
When the plants were exposed to 4oC, photosynthetic efficiency decreased in all five accessions while
NPQ increased (Figure 7). NPQ and qI showed the highest variation in the initial response to low
temperature, but IT was consistently lower than all other accessions in both parameters. In response to
increases in light intensity, IT and SW had increased qI and NPQ values less than Col-0, WS-2, and Ler.

Natural Variation in the Acclimation of Photosynthesis to Low Temperatures
After three weeks at 4oC, all five accessions were showing photosynthetic acclimation to low
temperatures (Figure 8). II increased over initial cold values and quenching parameters were lower. IT

22

Figure 6 Fluorescence Parameters of Five Arabidopsis Accessions
II (A), NPQ (B), qE (C), and qI (D) of SW (black lines), IT (red lines), Col-0 (green
lines), Ler (blue lines), and WS-2 (cyan lines). The light regime is shown on the righthand axis (dotted black line). n=3 for all accessions except for Col-0 n=2

23

Figure 7 Immediate Response of Fluorescence Parameters of Five Arabidopsis Accessions to a
Decrease in Temperature
II (A), NPQ (B), qE (C), and qI (D) of SW (black lines), IT (red lines), Col-0 (green
lines), Ler (blue lines), and WS-2 (cyan lines). The light regime is shown on the righthand axis (dotted black line). The temperature decreased from 22oC to 4oC at 13:00. n=3
for all accessions except for Col-0 n=2

24

Figure 8 Acclimation Response of Fluorescence Parameters of Five Arabidopsis Accessions after
Three Weeks at 4oC
II (A), NPQ (B), qE (C), and qI (D) of SW (black lines), IT (red lines), Col-0 (green
lines), Ler (blue lines), and WS-2 (cyan lines). The light regime is shown on the righthand axis (dotted black line). n=3 for all accessions except for Col-0 n=2

25

still had lower NPQ and qI, but there was little variation between the other accessions in these parameters.
qE varied only at higher light intensities, where IT and SW were low and Col-0, WS-2, and Ler were high.
II was highest in Ler and lowest in SW, except at higher light intensities where IT dropped below SW.

There is Little Natural Variation in Fluorescence Parameters after Cold Treatment
After 38 days at 4oC, plants were returned to 22oC. All five accessions showed improved photosynthetic
efficiency and decreased non-photochemical quenching upon return to 22oC (Figure 9). There was little
variation in II, NPQ, qE, or qI. The only exception was qE in WS-2, which was higher than all the other
accessions at high light.

FV/FM is Highly Variable between Accessions of Arabidopsis at Low Temperature
There was very little variation in FV/FM prior to cold treatment (Figure 10). The day after cold treatment,
FV/FM decreased in all accessions measured, but to varying levels; IT and WS-2 decreased the most.
Accessions began acclimating at varying times, with Ler and Col-0 being the earliest at around five days
and IT being the slowest at around 15 days. After 38 days in the cold, Ler had recovered to around 97%
of the warm FV/FM value, while IT had only recovered to about 87%.

26

Figure 9 Post-cold Fluorescence Parameters of Five Arabidopsis Accessions
II (A), NPQ (B), qE (C), and qI (D) of SW (black lines), IT (red lines), Col-0 (green
lines), Ler (blue lines), and WS-2 (cyan lines). The light regime is shown on the righthand axis (dotted black line). n=3 for all accessions except for Col-0 n=2

27

Figure 10 Acclimation of FV/FM to Low Temperature in Five Accessions of Arabidopsis
FV/FM was measured at 22oC, during 38 days at 4oC, and after plants were returned to
22oC for several days. SW (black lines), IT (red lines), Col-0 (green lines), Ler (blue
lines), and WS-2 (cyan lines), n=3 for all accessions except for Col-0 n=2

28

Discussion
Using the DEPI system, I measured chlorophyll fluorescence parameters simultaneously in five
accessions of Arabidopsis. I measured FV/FM, II, NPQ, qE, and qI at 22oC, at 4oC for 38 days, and after
returning plants to 22oC. All the plants acclimated to low temperatures, but there was variation between
the different accessions. The variation was largest for the FV/FM parameter, which would be the best
phenotype to use for a screen to identify the genetic basis of the variation based off this experiment.

There was an almost 20% difference between the FV/FM values of two ecotypes included in this study:
SW and IT. This difference may be large enough to use the existing RIL population between SW and IT
to screen for QTL underlying this phenotype. There are already approximately 400 genotyped
recombinant inbred lines between SW and IT (Grillo et al., 2013). The DEPI system will allow one to
screen the RIL lines for differences in FV/FM in the cold in a high throughput manner, as well as looking
at the other fluorescence parameters. This kind of high-throughput screen has not been possible
previously, and it could open up a whole new genetic resource for studying photosynthetic response to the
environment.

The results also raise the possibility of using the DEPI system to perform GWAS studies to identify QTL
associated with photosynthetic acclimation to low temperatures. Two days after the temperature was
lowered to 4oC, there was more than a 10% difference between FV/FM values in Ler and IT. After
approximately two weeks in the cold, the difference was more than 25% between them. This is a large
difference in FV/FM. However, the possibility of using these data for a GWAS study is limited. Five
accessions are not enough to project what kind of success a GWAS study might have. One would need to
screen a larger number of accessions before deciding to perform a GWAS study or deciding exactly
which parameters to use. One could also be more deliberate in the choice of accessions for this kind of
experiment. IT and SW were chosen because there is an existing resource between the two and they
represent the southern and northern extremes of the natural Arabidopsis range. Col-0, Ler, and WS were
29

chosen simply because their native range was between IT and SW. If one chose accessions from different
native environments, with different winter conditions, with different freezing tolerances, etc., one might
expect a larger natural difference in these fluorescence parameters. The results presented here show that
differences in fluorescence parameters can be detected in just using a very small number of accessions.

Both RIL studies and GWAS will only identify the QTLs underlying photosynthetic acclimation to low
temperatures. Further work would then need to be done to narrow the QTLs down to identify the
individual genes affecting photosynthetic acclimation. The DEPI system and the ability to perform these
large screens for differences in photosynthetic acclimation to low temperatures is an important first step
towards identifying genes involved in photosynthetic acclimation to low temperatures.

30

Materials and Methods
Plant Materials
Five accessions of Arabidopsis thaliana (SW, IT, Col-0, WS, and Ler) were grown in soil at 22oC under
around 120 mol photons m-2 s-1 for eight hour days. Three plants of each accession were planted, but
only two Col-0 germinated. After three weeks, plants were transferred to the DEPI chamber under the
same conditions. Light intensity changed to 250 mol photons m-2 s-1 at 14:00, 500 mol photons m-2 s-1
at 15:00, and 750 mol photons m-2 s-1 at 16:00 for the duration of the experiment. After four days, the
temperature was dropped to 4oC at 13:00. Plants remained at 4oC for 38 days, and then the temperature
was raised back to 22oC at 13:00 for the remainder of the experiment.

Fluorescence Measurements
Images were taken twice during the 120 mol photons m-2 s-1 light, then once after about 1 hour at each
light intensity. Fluorescence calculations were made using the Visual Phenomics software according to
established methods (Baker, 2007; Genty, 1989).
FV/FM = (Fm-F0)/Fm
II = (Fm’-Fs)/Fm’
NPQ = (Fm-Fm’)/Fm’
qE = (Fm’’-Fm’)/Fm’
qI = (Fm-Fm’’)/Fm’’

31

REFERENCES

32

REFERENCES

Alonso-Blanco, C. and Koornneef, M. (2000) Naturally occurring variation in Arabidopsis: an
underexploited resource for plant genetics. Trends in Plant Science, 5, 22-29.
Atwell, S., Huang, Y.S., Vilhjalmsson, B.J., Willems, G., Horton, M., Li, Y., Meng, D., Platt, A., Tarone,
A.M., Hu, T.T. and Nordborg, M. (2010) Genome-wide association study of 107 phenotypes in
Arabidopsis thaliana inbred lines. Nature, 465, 627-631.
Baker, N.R., Harbinson, J. and Kramer, D.M. (2007) Determining the limitations and regulation of
photosynthetic energy transduction in leaves. Plant, Cell and Environment, 30, 1107-1125.
Beavis, W.D. (1998) QTL analyses: power, precision, and accuracy. Molecular Dissection of Complex
Traits, 145-162.
Flood, P.J., Harbinson, J. and Aarts, M.G.M. (2011) Natural genetic variation in plant photosynthesis.
Trends in Plant Science, 16, 327-335.
Genty, B., Briantais, J.M. and Baker, N.R. (1989) The relationship between the quantum yield of
photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica
Acta. 990, 87-92.
Grillo, M.A., Li, C., Hammond, M., Wang, L, and Schemske, D.W. (2013) Genetic architecture of
flowering time differentiation between locally adapted populations of Arabidopsis thaliana. New
Phytologist, 197, 1321-1331.
Jung, H.S. and Niyogi, K.K. (2009) Quantitative Genetic Analysis of Thermal Dissipation in Arabidopsis.
Plant Physiol. 150, 977-986.
Mauricio, R. (2001) Mapping Quantitative Trait Loci in Plants: Uses and Caveats for Evolutionary
Biology. Nature Reviews, 2, 370-381.
Oakley, C. G., Ågren, J., Atchison, R.A. and Schemske, D.W. (2014) QTL mapping of freezing tolerance:
links to fitness and adaptive trade-offs. Molecular Ecology, Epub.
Öquist, G., Hurry, V.M. and Huner, N.P.A. (1993) Low-Temperature Effects on Photosynthesis and
Correlation with Freezing Tolerance in Spring and Winter Cultivars of Wheat and Rye. Plant Physiol.
101, 245-250.

33

CHAPTER 3: MUTANT SCREENS FOR VARIATION IN PHOTOSYNTHETIC ACCLIMATION TO
LOW TEMPERATURE
Introduction
Photosynthetic acclimation to low temperature is an important process for plants to grow and develop
under prolonged exposure to cold. Photosynthetic acclimation is positively correlated with cold tolerance
i.e. plants that achieve the highest levels of photosynthetic acclimation also reach the highest levels of
cold tolerance (Öquist et al., 1993).

Maximum cold tolerance relies on cold acclimation, the ability to increase freezing tolerance when
exposed to low, non-freezing temperatures (Thomashow, 1999). Cold acclimation is accompanied by
changes in gene expression as well as metabolic and structural changes in the plant (Thomashow, 1999).
Much work has been done to understand the importance of changes in levels of various genes at low
temperature. Many transcription factors are associated with the reprogramming of the transcriptome in the
cold (Fowler et al., 2002; Lee et al., 2005). Mutants with altered expression levels of some of these
transcription factors show large affects on cold acclimation. For instance, overexpression of CBF1, CBF2,
and CBF3, each of which is induced rapidly in response to low temperature and encodes an AP2/ERF
family transcription factor, leads to expression of cold-regulated (COR) genes and increased freezing
tolerance without exposing plants to low temperature (Jaglo-Ottosen et al., 1998).

Here I tested whether overexpression of MYB44, CRF3, DEAR1, ERF5, and HSFC1 affected
photosynthetic acclimation to low temperature. These genes were chosen as each is rapidly induced in
response to low temperature in parallel with CBF1, CBF2, and CBF3 and encodes a transcription factor.
It is possible that these transcription factors affect photosynthetic acclimation because there is a positive
correlation between cold tolerance and photosynthetic acclimation.

34

I also tested the effects of ZAT10, HB12, AS1, PMZ/SAP12, TCP4, and RAP2.6L on photosynthetic
acclimation to low temperature as each of these proteins has been associated with stress tolerance: ZAT10
is C2H2-EAR zinc finger transcription factor which elevates the expression of reactive oxygen-defense
transcripts (Mittler et al., 2006); HB12 is a homeodomain-leucine zipper TF which controls development
and water-stress response early in Arabidopsis development (Re et al., 2014); AS1 is a MYB domain TF
which functions in leaf development and plant immune responses to biotic stress (Nurmberg et al., 2007);
PMZ/SAP12 is a stress-associated protein with redox-dependent function in plants, potentially
functioning as a redox sensor for other TFs (Stroher et al., 2009); TCP4 is a transcription factor that
regulates leaf morphogenesis and interacts with auxin, gibberellic acid, and abscisic acid (Sarvepalli et
al., 2011); RAP2.6L is an AP2/ERF TF that functions in stomatal closure and antioxidant gene transcript
induction (Liu et al., 2012).

Finally, I tested the role of the CAMTA1, CAMTA2, and CAMTA3 transcription factors on
photosynthetic acclimation to low temperature as these genes function in the rapid induction of CBF1,
CBF2, and CBF3 (Kim et al., 2013). In addition, they regulate the expression of genes associated with
photosynthesis (Kim et al., 2013).

35

Results
Identification of Transcription Factors that Affect Chlorophyll Fluorescence Parameters at Warm
Temperatures and in the Cold
I used the DEPI system to measure photosynthetic acclimation to low temperatures in five Arabidopsis
transgenic lines that overexpress transcription factors (TFs) that have been found to be upregulated early
in response to low temperature. These TFs were CRF3, DEAR1, ERF5, HSFC1, and MYB44. The wildtype accession of Arabidopsis that was transformed to overexpress these TFs was Col-0. Plants
overexpressing MYB44 showed very little difference in photosynthetic efficiency when compared to the
wild-type plants (Figure 11). One day after the temperature was lowered from 22oC to 4oC, II was
slightly higher in MYB44 plants than in wild-type plants, but before the cold and after three weeks at 4oC
it was about the same as the Col-0 plants. The CRF3 overexpressing plants also showed slightly higher II
when the temperature was lowered, but after three weeks in the cold the CRF3 plants had a lower
photosynthetic efficiency than the wild-type plants. Plants overexpressing DEAR1 had a higher II than
Col-0 plants at 22oC, as well as during the cold treatment. ERF5 overexpressing plants showed no
difference in photosynthetic efficiency at 22oC or at 4oC. Like the DEAR1 plants, plants overexpressing
HSFC1 had a higher II than wild-type plants before the cold treatment. In addition, the HSFC1 plants had
the highest II values of any of the overexpression lines, both one day and three weeks after the plants
were exposed to low temperatures.

At 22oC, there was little difference in non-photosynthetic quenching at low light, but at higher light
intensities NPQ was slightly lower in the MYB44 plants when compared to Col-0 plants (Figure 12). One
day after plants were exposed to low temperatures, the MYB44 plants had a slightly higher NPQ at the
higher light intensities, but after three weeks there was no difference between the MYB44 and Col-0
plants. Non-photochemical quenching was lower in the CRF3 plants than in the wild-type plants at both
22oC and 4oC, although one day after the temperature change there was no difference at higher light

36

Figure 11 II in Cold Acclimation Associated Transcription Factors at Three Time Points
II in wild-type plants and five mutants: MYB44, CRF3, DEAR1, ERF5, and HSFC1 at
warm temperatures (left block), after one day in the cold (center block), and after three
weeks in the cold (right block). Each column within a block represents one measurement.
The scale bar represents the range of II values. Light intensity increased at the end of
each day.
37

Figure 12 NPQ in Cold Acclimation Associated Transcription Factors at Three Time Points
NPQ in wild-type plants and five mutants: MYB44, CRF3, DEAR1, ERF5, and HSFC1
at warm temperatures (left block), after one day in the cold (center block), and after three
weeks in the cold (right block). Each column within a block represents one measurement.
The scale bar represents the range of NPQ values. Light intensity increased at the end of
each day.
38

intensities. DEAR1 plants varied from Col-0 plants only at low light intensities the day after the
temperature changed to 4oC, where NPQ was lower. There was no difference in non-photochemical
quenching between the ERF5 plants and wild-type plants throughout the experiment. NPQ was slightly
lower in the HSFC1 plants at high light intensities and 22oC, but slightly higher at high light intensities
shortly after the temperature change, though lower at 4oC and low light intensities.

For the two components of NPQ, qE and qI, there was only slight variation between the MYB44 plants
and Col-0 plants at 22oC or 4oC (Figure 13). qE in the CRF3 plants was slightly lower early in the day at
both temperatures, and qI was lower as well. DEAR1 plants had a slightly lower qE at high light
intensities, but not much difference in qI. The ERF5 plants showed no difference from wild-type plants in
either parameter, but qE in HSFC1 plants was lower than in the wild-type plants, except at low light
intensities after three weeks in the cold, where it was higher. qI was lower at low light intensities in the
HSFC1 plants, but the same at high light intensities except early in the cold, where HSFC1 had higher qI.

Differences in Photosynthetic Properties in Transcription Factor Mutants and Overexpression Lines at
22oC and 4oC
II in ZAT10 overexpression lines, HB12 overexpression lines, TCP4 overexpression lines, ZAT10
knockout mutant plants, PMZ knockout mutant plants, and RAP2.6L knockout mutant plants showed very
little difference when compared to Col-0 plants, but the AS1 overexpression lines were different (Figure
14). At 22oC, AS1 plants showed no difference, but II was clearly lower in the AS1 plants at 4oC, even
after two weeks of acclimation. Only pmz plants, zat10 plants, and the AS1 plants showed a difference in
NPQ when compared to the wild-type plants (Figure 14). The pmz plants and zat10 plants both had higher
NPQ at 22oC and 4oC. AS1 plants had lower NPQ than Col-0 plants at 22oC and after two weeks in the
cold, but not right after changing the temperature to 4oC. As in NPQ, qE was slightly higher in pmz plants
and zat10 plants, and slightly lower in the AS1 plants (Figure 14). There was no difference in the other

39

Figure 13 qE and qI in Cold Acclimation Associated Transcription Factors at Three Time Points
qE (left) and qI (right) in wild-type plants and five mutants: MYB44, CRF3, DEAR1,
ERF5, and HSFC1 at warm temperatures (left block), after one day in the cold (center
block), and after three weeks in the cold (right block). Each column within a block
represents one measurement. The scale bar represents the range of values for each
parameter. Light intensity increased at the end of each day.

40

Figure 14 Photosynthetic Parameters in Seven Arabidopsis Mutants at Three Time Points
II (top left), NPQ (top right), qE (bottom left), and qI (bottom right) in mutants and wild
type at 22oC (left block), one day after changing to 4oC (center block), and after two
weeks at 4oC (right block). Each column within a block represents one measurement the
day. The scale bar represents the range of values for each parameter.
41

overexpression and mutant lines. There was very little difference in qI in any line (Figure 14).

CAMTA Mutants Show Differences in Fluorescence Parameters at Warm Temperatures and in the
Cold
I tested a camta1/camta2/camta3/sid2-1 quadruple knockout mutant of Arabidopsis to determine if the
CAMTAs affect photosynthetic acclimation to low temperatures. I also screened Arabidopsis plants
carrying the sid2-1 mutation, which results in plants that are deficient in salicylic acid (SA) biosynthesis.
In addition, I tested a quadruple mutant of Arabidopsis carrying camta1/2/3/sid2-1 to test the contribution
of elevated SA levels in the camta triple mutant plants to any affects on fluorescence parameters.
Arabidopsis Col-0 plants were the background for these mutants. I screened these mutants under a
consistent, low light intensity, a sinusoidal day increasing to 1000 mol photons m-2 s-1, and a sinusoidal
day with light fluctuations increasing to approximately 1500 mol photons m-2 s-1.

With an unchanging, about 200 mol photons m-2 s-1 light during the day, II was about the same in col-0
plants, sid2-1 plants, and camta1/2/3 plants at 22oC and 4oC, but was lower in the camta1/2/3/sid2-1
plants at both temperatures (Figure 15). NPQ in the sid2-1 plants and the Col-0 plants was about the same
compared to each other at 22oC and 4oC (Figure 15). camta1/2/3 plants were lower before the cold and
just after the decrease in temperature, but were about the same after a week in the cold. When the
temperature went back to 22oC, NPQ was lower again in the camta1/2/3 plants. In the quadruple mutant
plants, NPQ showed little change at all temperatures, except it was higher at the end of the day right after
the cold started. qE was about the same for Col-0 plants and sid2-1 plants at 22oC, but slightly lower in
sid2-1 plants right after the temperature went to 4oC (Figure 15). It was slightly higher in the sid2-1 plants
after one week. camta1/2/3 plants had lower qE than wild-type plants at 22oC, but slightly higher at 4oC.
qE was higher in the camta1/2/3/sid2-1 plants at 22oC, but lower just after the temperature changed. After
one week, qE values were about the same between Col-0 plants and the quadruple mutant plants. qI values

42

Figure 15 Photosynthetic Parameters in CAMTA Mutants under Consistent Light at Four Time
Points
II (top left), NPQ (top right), qE (bottom left), and qI (bottom right) in CAMTA mutants
compared to wild type at warm temperatures (first block), after one day in the cold
(second block), after one week in the cold (third block), and after being returned to warm
temperatures (fourth block). Each column within a block represents one measurement.
The scale bar represents the range of values for each parameter.
43

were lower in both the camta1/2/3 plants at 22oC (Figure 15). sid2-1 plants had slightly higher qI values
before the cold treatment, but slightly lower qI values by the end of the day after the cold. At 4oC, qI
values were about the same in the Col-0 plants, the sid2-1 plants, and the camta1/2/3/sid2-1 plants, but
were slightly lower in camta1/2/3 plants.

Under a sinusoidal light regime during the day, sid2-1 plants and camta1/2/3 plants had about the same II
as wild-type plants before cold treatment (Figure 16). After the cold treatment, the sid2-1 plants and the
Col-0 plants showed little difference, but camta1/2/3 plants were lower. At 4oC, sid2-1 plants were
slightly higher and camta1/2/3 plants were slightly lower than wild-type plants at higher light intensities.
camta1/2/3/sid2-1 plants were lower than Col-0 plants at both 22oC and 4oC. FV/FM was about the same in
all the lines (Figure 16). Before the change in temperature, NPQ was about the same in wild-type plants
and the quadruple mutant plants (Figure 16). NPQ in sid2-1 plants was a little higher at high light
intensity, and in the camta1/2/3 plants it was a little lower. After the cold treatment NPQ was about the
same in all the lines. NPQ in the sid2-1 and Col-0 plants was the same at 4oC, and was slightly lower in
the camta1/2/3 plants just after the temperature changed. camta1/2/3/sid2-1 plants had a slightly lower
NPQ at 4oC, but it was induced later than in the wild-type plants. qE in the sid2-1 plants was only slightly
higher than wild-type plants before the change in temperature. In camata1/2/3 plants, qE was slightly
higher before the cold treatment and even higher just after the change to 4oC (Figure 16). After one week,
qE was still higher, but after returning to 22oC it was about the same as in Col-0 plants. The
camta1/2/3/sid2-1 plants had higher qE at 22oC before the temperature change, but a lower qE at 4oC.
After the temperature was raised to 22oC, qE was the same between Col-0 plants and the quadruple mutant
plants. qI was slightly higher before the cold treatment in the sid2-1 plants, but was the same as in the
wild-type plants once the temperature changed. camta1/2/3 plants had a lower qI than Col-0 plants before
the cold and at 4oC, but after the cold treatment qI was about the same between the Col-0 plants and
camta1/2/3 plants (Figure 16). qI in camta1/2/3/sid2-1 plants was slightly lower than in wild-type plants
at 22oC, but about the same at 4oC.
44

Figure 16 Photosynthetic Parameters in CAMTA Mutants under Sinusoidal Light at Four Time
Points
II (top left), NPQ (top right), qE (bottom left), and qI (bottom right) in CAMTA mutants
compared to wild type at warm temperatures (first block), after one day in the cold
(second block), after one week in the cold (third block), and after being returned to warm
temperatures (fourth block). Each column within a block represents one measurement.
The scale bar represents the range of values for each parameter.
45

Under a sinusoidal light regime with fluctuating light intensity spikes, II was about the same between
Col-0 plants and sid2-1 plants (Figure 17). In the camta1/2/3 plants, II was slightly lower during the high
light spikes, but the same during the lower light periods at 22oC before cold and early at 4oC. After about
ten days, II was lower at all light intensities at 4oC, and also when returned to 22oC. In the quadruple
mutant plants, II was always lower. There was no obvious difference in FV/FM between any of the lines
(Figure 17). NPQ was slightly higher in the sid2-1 plants during high light spikes at 22oC and at 4oC when
compared to wild-type plants (Figure 17). In camta1/2/3 plants, NPQ was lower before the cold and early
in the cold treatment, but after approximately ten days in the cold and after returning to 22oC it was about
the same. NPQ was slightly lower in camta1/2/3/sid2-1 plants at 22oC compared to the Col-0 plants. At
4oC, the increase in NPQ was slightly delayed, and NPQ was slightly lower in the quadruple mutant
plants. qE in the sid2-1 plants was about the same in Col-0 plants and sid2-1 plants at both temperatures
(Figure 17). The camta1/2/3 plants had a lower qE during high light spikes before the cold, but a higher qE
during the lower light periods. Early in the cold treatment, qE was higher in the camta1/2/3 plants, but
after ten days it was about the same as in the wild-type plants. When returned to 22oC, qE was about the
same in the camta1/2/3 plants as well. In the quadruple mutant plants, qE was lower than in Col-0 plants.
qI was slightly higher in the sid2-1 plants before the cold treatment, but was about the same as wild-type
plants after that (Figure 17). camta1/2/3 plants had a lower qI before and through the cold treatment, but
after the cold treatment it was about the same as in Col-0 plants. qI was induced slightly more slowly in
the camta1/2/3/sid2-1 plants, but it was about the same as wild-type plants at 22oC and early in the cold.
After about ten days in the cold it was slightly lower than qI in Col-0 plants.

46

Figure 17 Photosynthetic Parameters in CAMTA Mutants under Fluctuating Light at Four Time
Points
II (top left), NPQ (top right), qE (bottom left), and qI (bottom right) in CAMTA mutants
compared to wild type at warm temperatures (first block), after one day in the cold
(second block), after one week in the cold (third block), and after being returned to warm
temperatures (fourth block). Each column within a block represents one measurement.
The scale bar represents the range of values for each parameter.
47

Discussion
The experiments described here identified several Arabidopsis mutants with altered photosynthetic
acclimation to low temperatures, as well as some with altered photosynthesis when plants were grown at
normal warm growth temperatures.

Several of the genes affected in the mutants are good candidates for involvement in photosynthetic
acclimation to low temperatures. HSFC1 overexpression affected photosynthesis, both at normal
temperatures and at low temperatures. The HSFC1 overexpression line would be interesting to study
further because despite having higher photosynthetic efficiency it grows much smaller than the wild type.
Overexpressing HSFC1 appears to confer higher photosynthetic efficiency, but HSFC1 overexpression
plants do not grow well as one might expect. DEAR1 overexpression plants also show some differences
in photosynthetic efficiency at 22oC and 4oC, so it may also be involved in photosynthesis or
photosynthetic acclimation. CRF3 overexpression plants would be interesting to investigate further as
well, since they had higher II immediately after being transferred to the cold, but after three weeks they
had a lower II than the Col-0 plants. This overexperssion line seems better able to handle the cold
initially, but is unable to acclimate photosynthetically as well as wild-type plants. However, this
experiment did have an extra high light stress that may have affected the results. Measurements were
made every five minutes during the day, which exposes the plants to many saturating pulses. Too many
measurements too close together affect photosynthesis, de

II

under constant light and decreasing

FV/FM over time. This experiment should be done again with fewer measurements to confirm the
phenotypes seen here.

AS1 overexpression plants are severely inhibited photosynthetically at low temperatures. This
overexpression line does poorly when the temperature is decreased to 4oC, and is never able to acclimate
photosynthesis to low temperatures. AS1 is a developmental transcription factor involved in the

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symmetrical development of leaves. Its role in photosynthesis is unclear, but appears to be significant at
low temperatures. AS1 is known to interact with several other proteins during development, and it may
interact with proteins for regulating photosynthesis in the cold as well. Young leaves, especially those
developed in the cold, have a higher photosynthetic efficiency at low temperatures and when returned to
warm temperatures. It is possible that this is due to some sort of acclimation which occurs during the
development of the leaves. If AS1 is a negative controller of this developmental acclimation, then
overexpressing AS1 would inhibit the acclimation of these plants, which appears to be what happened in
these overexpression lines.

camta1/2/3/sid2-1 plants are an interesting candidate for further study as well. This mutant had FV/FM
values comparable to wild-type plants throughout the experiment, but II was lower at all temperatures
and under all the light conditions tested. Alone, neither sid2-1 plants nor camta1/2/3 plants had similar II
values. In the camta1/2/3 plants SA levels are elevated (Kim et al., 2013). It is possible that the increased
SA levels in the camta1/2/3 plants help the plant photosynthetically somehow, but when SA biosynthesis
is knocked out, the plant is unable to compensate and photosynthetic efficiency is reduced. Nonphotochemical quenching is also affected in these mutant plants. Induction of the NPQ response is
delayed in these mutants, particularly in the qI component, so that NPQ builds up later in these plants.
Further work would be needed to understand how CAMTA1, 2, and 3 affect photosynthesis and how SA
helps to make up for a lack of CAMTA gene products.

Photosynthetic acclimation to low temperature is a complicated and highly regulated process. Gene
changes are sure to play an important role in the acclimation process, and identifying the genes involved
in acclimation will allow us to better understand the mechanisms of acclimation. It will also open the path
to improving photosynthesis at low temperatures in plants. I have identified several candidate genes for
future study for their involvement in photosynthetic acclimation to low temperature, and the DEPI system

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will allow larger screening of mutant populations to identify other potential genes in the photosynthetic
acclimation pathway.

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Materials and Methods
Plant Materials
All plants were grown in soil at around 22oC. MYB44, CRF3, DEAR1, ERF5, and HSFC1
overexpression lines were grown with day length of twelve hours under a light intensity of approximately
120 mol photons m-2 s-1, which increased to 250 mol photons m-2 s-1 at 17:00, 500 mol photons m-2 s-1
at 18:00, and 750 mol photons m-2 s-1 at 17:00. ZAT10, HB12, and AS1 overexpression lines and
PMZ/SAP12, TCP4, and RAP2.6L knockout mutants were grown with a day length of eight hours under a
sinusoidal light regime reaching around 1500 mol photons m-2 s-1 at midday. CAMTA mutants and
SID2-1 mutants were grown with an eight hour day length under a sinusoidal light regime reaching about
500 mol photons m-2 s-1 at midday. After approximately three weeks, measurements began and the light
regime for these mutants changed. They went onto a three day cycle with the first day consistent light at
approximately 120 mol photons m-2 s-1, the second sinusoidal light reaching around 500 mol photons
m-2 s-1 at midday, and the third day sinusoidal light reaching about 500 mol photons m-2 s-1 at midday
with high light fluctuations reaching approximately 1000 mol photons m-2 s-1 at midday (Figure 18).

Fluorescence Measurements
For the first overexpression experiment (MYB44, etc.), II images were taken every ten minutes during
the day. qE/qI images were taken twice at around 120 mol photons m-2 s-1, and then once after about 1
hour at each higher light intensity. For the second overexpression/mutant experiment (ZAT10, etc), FV/FM
images were taken just prior to lights coming on. II images were taken thirty minutes after the lights
turned on, and then every sixty minutes after that during the day. qE/qI images were taken ten minutes
after each II image. For the CAMTA mutants, FV/FM images were taken just prior to lights coming on.
On the consistent light day, II images were taken three times during the day, and qE/qI images were taken
four minutes after each of the first two II images. On the sinusoidal day, II images were taken every
thirty minutes during the day, and qE/qI images were taken four minutes after the first II image, and every
51

Figure 18 Fluctuating Day Light Intensities for camta Experiment
Light intensities at each time of day for the fluctuating light day of the camta experiment.

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other one after that. On the fluctuating light day, II images were taken at every light intensity, and qE/qI
images were taken after the first II image and after the fluctuation which followed it, as well as every
other light intensity and fluctuation after that. Calculations were made using the Visual Phenomic
program according to established protocols (Baker, 2007; Genty, 1989).
II = (Fm’-Fs)/Fm’
NPQ = (Fm-Fm’)/Fm’
qE = (Fm’’-Fm’)/Fm’
qI = (Fm-Fm’’)/Fm’’

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REFERENCES

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REFERENCES

Baker, N.R., Harbinson, J. and Kramer, D.M. (2007) Determining the limitations and regulation of
photosynthetic energy transduction in leaves. Plant, Cell and Environment, 30, 1107-1125.
Fowler, S. and Thomashow, M.F. (2002) Arabidopsis Transcriptome Profiling Indicates That Multiple
Regulatory Pathways Are Activated during Cold Acclimation in Addition to the CBF Cold Response
Pathway. The Plant Cell, 14, 1675-1690.
Genty, B., Briantais, J.M. and Baker, N.R. (1989) The relationship between the quantum yield of
photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica
Acta. 990, 87-92.
Jaglo-Ottosen, K.R., Gilmour, S.J., Zarka, D.G., Schabengerger, O. and Thomashow, M.F. (1998)
Arabidopsis CBF1 overespression induces COR genes and enhances freezing tolerance. Science, 280,
104-106.
Kim, Y.S., Park, S., Gilmour, S.J. and Thomashow, M.F. (2013) Roles of CAMTA transcription factors
and salicylic acid in configuring the low-temperature transcriptome and freezing tolerance of Arabidopsis.
The Plant Journal, 75, 364-376.
Lee, B., Henderson, D.A. and Zhu, J. (2005) The Arabidopsis Cold-Responsive Transcriptome and Its
Regulation by ICE1. The Plant Cell, 17, 3155-3175.
Liu, P., Sun, F., Gao, R. and Dong, H. (2012) RAP2.6L overexpression delays waterlogging induced
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Mittler, R., Kim, Y.S., Song, L., Coutu, J., Coutu, A., Ciftci-Yilmaz, S., Lee, H., Stevenson, B. and Zhu,
J. (2006) Gain- and loss-of-function mutations in Zat10 enhance the tolerance of plants to abiotic stress.
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Nurmberg, P.L, Knox, K.A., Yun, B., Morris, P.C., Shaftel, R., Hudson, A. and Loake, G.J. (2007) The
developmental selector AS1 is an evolutionarily conserved regulator of the plant immune response. PNAS,
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Öquist, G., Hurry, V.M. and Huner, N.P.A. (1993) Low-Temperature Effects on Photosynthesis and
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Stroher, E., Wang, X., Roloff, N., Klein, P., Husemann, A. and Dietz, K. (2009) Redox-Dependent
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Mechanisms. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 571-599.

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CHAPTER 4: OVERALL CONCLUSIONS
In conclusion, the development of new tools for measuring chlorophyll fluorescence under varying
conditions has opened up a number of possiblities for identifying genes involved in photosynthetic
acclimation to low temepratures. The DEPI system enables high-throughtput and continual measurements
under dynamic environmental conditions.

High-throughput fluorescence measurements are necessary to fully utilize many genetic approaches, such
as QTL mapping and mutant screens. When the DEPI system was first created, we could only measure
approximately thirty-two plants simultaneously. Now the DEPI systmes is capable of measuring more
than two hundred plants simultaneously. This makes screening RIL populations consisting of hundreds of
lines feasible, which will allow QTL mapping for various photosynthetic traits. This kind of highthroughput abiliy also makes it possible to screen many mutants at once to identify those with altered
photosynthetic acclimation to low temperatures.

The DEPI system also has improved our ability to monitor plants continuously over long periods of time
without disturbing the plants. This is critical for accurately measuring acclimation to dynamic
environmental conditions, such as temperature and light, over both narrow and broad time ranges. Now,
using the DEPI system, we can measure the rapid response of plants to a decrease in temperature, as well
as the long-term response of the same plants to extended exposure to low temperatures. When the DEPI
chambers were first built this was always the goal of the system, but there were many problems.
Hardware would crash and software would malfunction. Over time, we were able to identify many ‘bugs’
and problems with the system, and the engineers were able to overcome these problems. Now, the
stability of the system is greatly improved, and we are able to make these continuous observations for
several weeks in a row.

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The DEPI system also gives dynamic control over many variables. Once again this allows one to look at
the affects of the variable being tested while isolating it from other variables which could confound
interpretation of the results. Befor the DEPI systems, we had to grow plants in a chamber, and then move
them for measurement or to different chambers with different conditions. Now, we are able to grow plants
in the DEPI chambers, then dynamically change variables like temperature and light intensity in order to
measure the affects of the variables without ever moving or touching the plants.

The DEPI system greatly increases our abiliity to identify genes involved in photosynthetic acclimation to
low temperatures. We are able to do high-throughput screens to identify natural variation between
accessions of Arabidopsis, as well as mutants with altered photosynthetic acclimation to low
temperatures. We are able to make these measurements continuously, while we dynamically change the
environmental variables of the plants. The DEPI system is a valuable tool which has opened up many
genetic resources for use in the study of photosynthetic acclimation to low temperature.

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