Plant Physiology Preview. Published on February 10, 2017, as DOI:10.1104/pp.16.01767
1
Importance of fluctuations in light on plant photosynthetic acclimation
2 3
Silvere Vialet-Chabrand#, Jack S.A. Matthews#, Andrew J. Simkin, Christine A. Raines and Tracy Lawson*
4
School of Biological Sciences, University of Essex, Colchester, CO4 3SQ, UK.
5
# Joint 1st author
6
* Corresponding author: [email protected]
7 8 9
SVC, JSAM & TL designed the experiments; JSAM & SVC performed the measurements and; AJS & CAR carried out immunoblotting and associated analysis; and SVC, JSAM & TL analysed the data; all authors contributed to writing the MS.
10 11
SV-C was supported through a BBSRC grant BB/1001187_1 awarded to TL. JSAM is funding by a PhD NERC DTP studentship (Env-East DTP E14EE).
12 13
Short title: Plant responses to fluctuating light regimes
14 15
One-sentence summary: Fluctuating light influence acclimation in Arabidopsis thaliana independently of the light intensity
16 17
Abstract:
18
Acclimation of plants to light has been studied extensively, yet little is known about the effect of
19
dynamic fluctuations in light on plant phenotype and acclimatory responses. We mimicked natural
20
fluctuations in light over a diurnal period to examine the effect on photosynthetic processes and
21
growth of Arabidopsis thaliana. High and low light intensities, delivered via a realistic dynamic
22
fluctuating or square wave pattern were used to grow and assess plants. Plants subjected to square
23
wave light had thicker leaves and greater photosynthetic capacity compared with fluctuating light
24
grown plants. This together with elevated levels of proteins associated with electron transport
25
indicates greater investment in leaf structural components and photosynthetic processes. In
26
contrast plants grown under fluctuating light had thinner leaves, lower leaf light absorption but
27
maintained similar photosynthetic rates per unit leaf area to square wave grown plants. Despite high
28
light use efficiency plants grown under fluctuating light had a slow growth rate early in
29
development, likely due to the fact that plants grown under fluctuating conditions were not able to
30
fully utilize the light energy absorbed for carbon fixation. Diurnal leaf level measurements revealed a
31
negative feedback control of photosynthesis, resulting in a decrease in total diurnal carbon
32
assimilated of at least 20%. These findings highlight that growing plants under square wave growth
33
conditions ultimately fails to predict plant performance under realistic light regimes, and stresses
1 13, 2017 - Published by www.plantphysiol.org Downloaded from www.plantphysiol.org on February Copyright © 2017 American Society of Plant Biologists. All rights reserved. Copyright 2017 by the American Society of Plant Biologists
34
the importance of considering fluctuations in incident light in future experiments that aim to infer
35
plant productivity under natural conditions in the field.
36 37
Key words: Photosynthesis, Fluctuating Light, Dynamics, Acclimation, Diurnal.
2 13, 2017 - Published by www.plantphysiol.org Downloaded from www.plantphysiol.org on February Copyright © 2017 American Society of Plant Biologists. All rights reserved.
38 39
Abbreviations
40
[CO2] – Atmospheric CO2 concentration
41
A – Net CO2 assimilation rate per unit leaf area
42
A/Ci - Net CO2 assimilation rate (A) as a function of [CO2]
43
A/Q - Net CO2 assimilation rate (A) as a function of light intensity (Q)
44
Amass - Net CO2 assimilation rate per unit leaf dry mass
45
Asat – Light saturated assimilation rate
46
Ci – intercellular CO2 concentration
47
Cic – Ci at which limitation of A switches between Rubisco- and RuBP regeneration
48
Daily LUE – Daily light use efficiency
49
DFhigh - Measurements under diurnal high light fluctuating conditions
50
DFlow - Measurements under diurnal low light fluctuating conditions
51
FLH – Plants grown under fluctuating high light
52
FLL - Plants grown under fluctuating low light
53
Fq’/Fm’ – PSII operating efficiency
54
Fq’/Fv’ – PSII efficiency factor
55
Fv’/Fm’ – PSII maximum efficiency
56
gm - Mesophyll conductance
57
gs – Stomatal conductance to water vapour
58
Jmax - Maximum electron transport demand for RuBP regeneration
59
LUE – Light use efficiency
60
NPQ – Non-photochemical quenching
61
PPFD – Photosynthetic photon flux density
62
PSI – Photosystem I
63
PSII – Photosystem II
64
Q – Light intensity
65
Rday - Day respiration
66
Rdiurnal - Dark respiration measured at the beginning of the diurnal period
67
Rmodel - Dark respiration derived from A/Q curve analysis
68
SLA – Specific leaf area
69
SQH – Plants grown under square-wave high light
70
SQL – Plants grown under square-wave low light
71
Vcmax – Maximum rate of carboxylation by Rubisco
72
Γ – light-compensation point
3 13, 2017 - Published by www.plantphysiol.org Downloaded from www.plantphysiol.org on February Copyright © 2017 American Society of Plant Biologists. All rights reserved.
73 74
Introduction In the natural environment plants experience a range of light intensities and spectral
75
properties due to changes in sun angle and cloud cover in addition to shading from overlapping
76
leaves and neighbouring plants. Leaves are therefore subjected to spatial and temporal gradients in
77
incident light, which has major consequences for photosynthetic carbon assimilation (Pearcy, 1990;
78
Chazdon and Pearcy, 1991; Pearcy and Way, 2012). As light is the key resource for photosynthesis,
79
plants acclimate to the light environment under which they are grown to maintain performance and
80
fitness. Acclimation involves altering metabolic processes (incl. light harvesting and CO2 capture)
81
brought about by a range of mechanisms from adjustments to leaf morphology to changes in
82
photosynthetic apparatus stoichiometry (e.g. see, Athanasiou et al., 2010; Kono and Terashima
83
2014; Terashima et al., 2006) all of which impact on photosynthesis. The primary determinant of
84
crop yield is the cumulative rate of photosynthesis over the growing season, which is regulated by
85
the amount of light captured by the plant, and the ability of the plant to efficiently use this energy
86
for converting CO2 into biomass and harvestable yield (Sinclair and Muchow, 1999). Currently
87
considerable research efforts in plant biology focus on improving performance, including the plants’
88
ability to cope with changing abiotic or biotic factors in order to increase or maintain crop biomass
89
and yield to support the rising demands for food and fuel (Ort et al., 2015). Many current studies
90
employ transgenic approaches, and therefore plants are often grown in laboratory controlled
91
conditions (e.g. Lefebvre et al., 2005; Simkin et al., 2015; 2017; Kono and Terashima, 2016), however
92
with the ultimate aim to improve crops grown in the field (Rosenthal et al., 2011; Poorter et al.,
93
2016). Light is one of the most dynamic environmental factors that directly impacts on plant
94
performance, and it is therefore important to understand how plants acclimate to fluctuating light
95
environments such as those experienced under field conditions.
96
Plant acclimation to changes in irradiance can be categorised as (i) dynamic acclimation
97
which refers to a reversible biological process present within a given period of time (Walters and
98
Horton, 1994; Mullineaux, 2006; Okegawa et al., 2007; Athanasiou et al., 2010; Yin and Johnson,
99
2000; Tikkanen et al., 2010; Alter et al., 2012; Suorsa et al., 2012; Yamori, 2016); or (ii)
100
developmental acclimation which is defined as changes in morphology (e.g. leaf thickness and
101
density) resulting from a given growth light environment, and are largely irreversible (Weston et al.,
102
2000; Murchie, 2005), and is the focus of this study. The ability of plants to developmentally
103
acclimate to a given light environment is particularly well-demonstrated in leaves grown in sun and
104
shade conditions, which differ in photosynthetic efficiency, biochemistry (e.g. Rubisco content and
105
change in Photosystem II and I ratio), anatomy (e.g. chloroplast size and distribution) and
106
morphology (e.g. leaf mass area and thickness) (Givnish, 1988; Walters and Horton, 1994; Weston et
4 13, 2017 - Published by www.plantphysiol.org Downloaded from www.plantphysiol.org on February Copyright © 2017 American Society of Plant Biologists. All rights reserved.
107
al., 2000; Bailey et al., 2001; Bailey et al., 2004). Plants grown under high light intensity tend to
108
develop thicker leaves than those grown under low light intensity (Evans and Poorter, 2001), which
109
generally increases photosynthetic capacity per unit area improving the plant’s ability to utilize light
110
for carbon fixation (Terashima et al., 2006). Increased photosynthetic capacity is often strongly
111
correlated with the concentration of photosynthetic enzymes such as Rubisco, cytochrome f, H+-
112
ATPase and reaction centres (Foyer et al., 2012). Leaves acclimated to shade tend to have higher net
113
photosynthetic rates at lower light levels and a lower light compensation point compared to sun
114
leaves (Givnish 1988). To compare plants/species with different leaf thicknesses, previous studies
115
have used mass integrated photosynthesis as a proxy to assess the photosynthetic efficiency of
116
plants with different volumes of photosynthetic tissues (Garnier et al., 1999; Evans and Poorter,
117
2001; Wright et al., 2001). Previous studies investigating developmental acclimation have primarily
118
focused on the effect of light intensity, with less emphasis given to the effect of dynamic light during
119
growth, like that experienced under a natural environment. Fluctuations in light could have a
120
significant impact on acclimation processes during growth, and need to be investigated alongside
121
light intensity to assess the interaction between light regime and intensity.
122
Acclimation is the result of a balance between the cost of increasing leaf photosynthetic
123
capacity, which can be underutilized (Terashima et al., 2006; Oguchi et al., 2008), and the risk of
124
photo-oxidative damage if the mechanisms to dissipate excess energy received by the plant are not
125
sufficient (Li et al., 2009). Under natural environmental conditions, the random duration and
126
intensity of fluctuating light from passing clouds or leaf movements (sun- and shade-flecks) result in
127
incident light intensities below light saturation that reduce photosynthetic rates, whilst those
128
intensities greater than saturated lead to excess excitation energy that can result in short potential
129
“stress” periods and long term damage to leaf photosynthesis (Baker, 2008). Plants therefore
130
employ mechanisms that enable them to deal with these changes in excitation pressure, including
131
thermal dissipation of excitation energy. Such processes are termed non-photochemical quenching
132
(NPQ) and are mainly associated with changes in the xanthophyll cycle (Demmig-Adams and Adams,
133
1992; Muller et al., 2001) and protonation of PSII antenna proteins (Li et al., 2000; 2004) both of
134
which are linked to the proton gradient across the thylakoid membrane. Large diversity in light
135
acclimation exists between individuals and species (Murchie and Horton, 1997), partly due to the
136
random nature of light fluctuations and species-specific responses.
137
To date the majority of studies examining acclimation to fluctuating light conditions have
138
been carried out on plants grown under constant intensities of light and swapped to a simple light
139
pattern (consisting of one or more step changes in light intensity of different frequencies) (Yin and
140
Johnson, 2000; Tikkanen et al., 2010; Alter et al., 2012; Suorsa et al., 2012; Yamori, 2016). Under
5 13, 2017 - Published by www.plantphysiol.org Downloaded from www.plantphysiol.org on February Copyright © 2017 American Society of Plant Biologists. All rights reserved.
141
these light conditions, acclimation responses have often been monitored over a period of several
142
days (e.g. Alter et al., 2012; Athanasiou et al., 2010). Whilst this approach is powerful for studies on
143
the mechanisms of dynamic light acclimation, it fails to recognise the importance of how plants
144
developmentally acclimate to growth under fluctuating light intensities (Huxley, 1969), such as those
145
found in the natural field environment (Frechilla et al., 2004). There are only a handful of studies
146
that have examined the impact of “real” dynamic light environments on plant growth and
147
performance, and as far as we are aware none of these have used a controlled environment to
148
examine the direct impact of light. For example, Yamori (2016) revealed the importance of
149
unpredictable variations in environmental growth conditions (including light) that led to a reduction
150
in photosynthesis because plants were unable to fully acclimate to the highly dynamic variation in
151
light. However in this study, plants were grown under natural environmental conditions that
152
resulted in fluctuations in a number of environmental variables and therefore the impact of light
153
alone on the acclimation response could not be distinguished. Kulheim et al. (2002) compared field
154
grown non-photochemical quenching Arabidopsis mutants with those grown in controlled
155
environment chambers under constant or variable light intensity, and demonstrated that NPQ is
156
important for plant fitness in the field and under fluctuating environments reproduced in growth
157
chambers. However, when the plants were grown under constant light conditions no effect on plant
158
performance was observed, emphasizing the influence of growth environment on plant fitness.
159
Although the study by Kulheim et al. (2002) was one of the first to examine the influence of dynamic
160
and square wave growth light regimes on plant performance and growth, the dynamic light regime
161
used in the controlled environment did not mimic that observed in the field.
162
In order to fully understand how plants integrate fluctuations in incident light, and how this
163
influences acclimation and modifies plant growth, there is a need to grow plants in a controlled but
164
dynamic environment that mimics a light regime that would be experienced in the field. How plants
165
perform under these conditions and the differences in responses with those grown in square wave
166
light regimes is important, as models of steady state photosynthesis tend to overestimate
167
photosynthesis under fluctuating light regimes (Naumburg and Ellsworth, 2002). The importance of
168
developmental acclimation for plant performance has been demonstrated in “sun” (high light) and
169
“shade” (low light) leaves. However, it is not known if developmental acclimation to fluctuating light
170
intensity exists and if so how it may influence plant performance under dynamic light conditions,
171
such as those experienced in a natural environment, and force us to rethink experimental growth
172
conditions to draw conclusions on how plants will perform in the field. To address this, Arabidopsis
173
plants were grown and measured under fluctuating and non-fluctuating (or square wave) light
174
regimes at two different average intensities (high and low) (Fig. 1) and the performance of these
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175
plants and their ability to convert light energy into biomass evaluated.
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176
Results
177 178
Photo-acclimation of plants grown under different light regimes
179
Light response curves in which net CO2 assimilation rate (A) was measured as a function of
180
photosynthetic photon flux density (PPFD) (Q) (A/Q curves, Fig. 2A) displayed similar A values at
181
PPFDs below 250 µmol m-2 s-1 in plants grown under all the different light regimes: square wave high
182
(SQH) and low (SQL) and fluctuating high (FLH) and low (FLL) light intensity. Measurements of A at
183
PPFD above this value and light saturated assimilation rate (Asat, Table 1), were significantly greater
184
in plants grown under high light intensity compared with those grown under low light,
185
independently of the light regime (Fig.2A).
186
Generally, photosynthesis is measured per unit leaf area, however this area also represents a
187
volume of photosynthetic tissues that can differ among plants (e.g. different leaf thickness). To take
188
into consideration photosynthesis per unit leaf volume, we integrated A by mass of dry leaf (Amass).
189
There was significantly greater Amass in plants grown under fluctuating light regimes compared to
190
those grown under square wave light regimes (Fig. 2B), and as expected a tendency for plants grown
191
under high light regimes to have greater rates of Amass compared to plants grown under low light
192
regimes.
193
Dark respiration, derived from the A/Q curve (Rd-model) was significantly higher in plants grown under
194
SQH and there was a general tendency for higher respiration in plants grown in square wave light
195
intensity regimes compared with fluctuating regimes (Table. 1) in Rd-model as well as those measured
196
during diurnals (Rd-diurnal). However it should be noted that, Rd-diurnal measured at the start of the
197
diurnal was lower than that Rd-model determined from the A/Q analysis. Plants grown under SQH also
198
had a significantly higher light compensation point (Γ) compared to plants grown under fluctuating
199
light regimes (Table 1).
200
The large differences observed in the response of A to PPFD between plants grown under low and
201
high light intensity, was less significant for Photosystem II (PSII) operating efficiency (Fq’/Fm’) (Fig.
202
2C). Response of Fq’/Fm’ to PPFD was mainly driven by changes in the PSII efficiency factor (Fq’/Fv’)
203
(Fig. 2D). Fq’/Fm’ was also affected, although to a lower extent, by the maximum efficiency of PSII
204
(Fv’/Fm’) which was higher in plants grown under high PPFD (Fig. 2E), with low values illustrating
205
greater non-photochemical quenching. In general plants grown under fluctuating regimes had a
206
higher Fv’/Fm’ compared with those grown under square wave, particularly when measured under
207
high PPFDs. Plants grown under SQL showed the lowest values in both quenching parameters: Fq’/Fv’
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208
and Fv’/Fm’. Non-photochemical quenching (NPQ) increased more rapidly at low light intensity in
209
plants grown under SQL compared to plants grown in the other lighting regimes, and in general NPQ
210
had a tendency to be lower in plants grown under fluctuating light (Fig. 2F).
9 13, 2017 - Published by www.plantphysiol.org Downloaded from www.plantphysiol.org on February Copyright © 2017 American Society of Plant Biologists. All rights reserved.
211 212
Leaf properties in plants acclimated to different light regimes
213
Leaf absorbance (measured after 28 days of growth) was significantly different between plants
214
grown in the different light regimes (P
1
Importance of fluctuations in light on plant photosynthetic acclimation
2 3
Silvere Vialet-Chabrand#, Jack S.A. Matthews#, Andrew J. Simkin, Christine A. Raines and Tracy Lawson*
4
School of Biological Sciences, University of Essex, Colchester, CO4 3SQ, UK.
5
# Joint 1st author
6
* Corresponding author: [email protected]
7 8 9
SVC, JSAM & TL designed the experiments; JSAM & SVC performed the measurements and; AJS & CAR carried out immunoblotting and associated analysis; and SVC, JSAM & TL analysed the data; all authors contributed to writing the MS.
10 11
SV-C was supported through a BBSRC grant BB/1001187_1 awarded to TL. JSAM is funding by a PhD NERC DTP studentship (Env-East DTP E14EE).
12 13
Short title: Plant responses to fluctuating light regimes
14 15
One-sentence summary: Fluctuating light influence acclimation in Arabidopsis thaliana independently of the light intensity
16 17
Abstract:
18
Acclimation of plants to light has been studied extensively, yet little is known about the effect of
19
dynamic fluctuations in light on plant phenotype and acclimatory responses. We mimicked natural
20
fluctuations in light over a diurnal period to examine the effect on photosynthetic processes and
21
growth of Arabidopsis thaliana. High and low light intensities, delivered via a realistic dynamic
22
fluctuating or square wave pattern were used to grow and assess plants. Plants subjected to square
23
wave light had thicker leaves and greater photosynthetic capacity compared with fluctuating light
24
grown plants. This together with elevated levels of proteins associated with electron transport
25
indicates greater investment in leaf structural components and photosynthetic processes. In
26
contrast plants grown under fluctuating light had thinner leaves, lower leaf light absorption but
27
maintained similar photosynthetic rates per unit leaf area to square wave grown plants. Despite high
28
light use efficiency plants grown under fluctuating light had a slow growth rate early in
29
development, likely due to the fact that plants grown under fluctuating conditions were not able to
30
fully utilize the light energy absorbed for carbon fixation. Diurnal leaf level measurements revealed a
31
negative feedback control of photosynthesis, resulting in a decrease in total diurnal carbon
32
assimilated of at least 20%. These findings highlight that growing plants under square wave growth
33
conditions ultimately fails to predict plant performance under realistic light regimes, and stresses
1 13, 2017 - Published by www.plantphysiol.org Downloaded from www.plantphysiol.org on February Copyright © 2017 American Society of Plant Biologists. All rights reserved. Copyright 2017 by the American Society of Plant Biologists
34
the importance of considering fluctuations in incident light in future experiments that aim to infer
35
plant productivity under natural conditions in the field.
36 37
Key words: Photosynthesis, Fluctuating Light, Dynamics, Acclimation, Diurnal.
2 13, 2017 - Published by www.plantphysiol.org Downloaded from www.plantphysiol.org on February Copyright © 2017 American Society of Plant Biologists. All rights reserved.
38 39
Abbreviations
40
[CO2] – Atmospheric CO2 concentration
41
A – Net CO2 assimilation rate per unit leaf area
42
A/Ci - Net CO2 assimilation rate (A) as a function of [CO2]
43
A/Q - Net CO2 assimilation rate (A) as a function of light intensity (Q)
44
Amass - Net CO2 assimilation rate per unit leaf dry mass
45
Asat – Light saturated assimilation rate
46
Ci – intercellular CO2 concentration
47
Cic – Ci at which limitation of A switches between Rubisco- and RuBP regeneration
48
Daily LUE – Daily light use efficiency
49
DFhigh - Measurements under diurnal high light fluctuating conditions
50
DFlow - Measurements under diurnal low light fluctuating conditions
51
FLH – Plants grown under fluctuating high light
52
FLL - Plants grown under fluctuating low light
53
Fq’/Fm’ – PSII operating efficiency
54
Fq’/Fv’ – PSII efficiency factor
55
Fv’/Fm’ – PSII maximum efficiency
56
gm - Mesophyll conductance
57
gs – Stomatal conductance to water vapour
58
Jmax - Maximum electron transport demand for RuBP regeneration
59
LUE – Light use efficiency
60
NPQ – Non-photochemical quenching
61
PPFD – Photosynthetic photon flux density
62
PSI – Photosystem I
63
PSII – Photosystem II
64
Q – Light intensity
65
Rday - Day respiration
66
Rdiurnal - Dark respiration measured at the beginning of the diurnal period
67
Rmodel - Dark respiration derived from A/Q curve analysis
68
SLA – Specific leaf area
69
SQH – Plants grown under square-wave high light
70
SQL – Plants grown under square-wave low light
71
Vcmax – Maximum rate of carboxylation by Rubisco
72
Γ – light-compensation point
3 13, 2017 - Published by www.plantphysiol.org Downloaded from www.plantphysiol.org on February Copyright © 2017 American Society of Plant Biologists. All rights reserved.
73 74
Introduction In the natural environment plants experience a range of light intensities and spectral
75
properties due to changes in sun angle and cloud cover in addition to shading from overlapping
76
leaves and neighbouring plants. Leaves are therefore subjected to spatial and temporal gradients in
77
incident light, which has major consequences for photosynthetic carbon assimilation (Pearcy, 1990;
78
Chazdon and Pearcy, 1991; Pearcy and Way, 2012). As light is the key resource for photosynthesis,
79
plants acclimate to the light environment under which they are grown to maintain performance and
80
fitness. Acclimation involves altering metabolic processes (incl. light harvesting and CO2 capture)
81
brought about by a range of mechanisms from adjustments to leaf morphology to changes in
82
photosynthetic apparatus stoichiometry (e.g. see, Athanasiou et al., 2010; Kono and Terashima
83
2014; Terashima et al., 2006) all of which impact on photosynthesis. The primary determinant of
84
crop yield is the cumulative rate of photosynthesis over the growing season, which is regulated by
85
the amount of light captured by the plant, and the ability of the plant to efficiently use this energy
86
for converting CO2 into biomass and harvestable yield (Sinclair and Muchow, 1999). Currently
87
considerable research efforts in plant biology focus on improving performance, including the plants’
88
ability to cope with changing abiotic or biotic factors in order to increase or maintain crop biomass
89
and yield to support the rising demands for food and fuel (Ort et al., 2015). Many current studies
90
employ transgenic approaches, and therefore plants are often grown in laboratory controlled
91
conditions (e.g. Lefebvre et al., 2005; Simkin et al., 2015; 2017; Kono and Terashima, 2016), however
92
with the ultimate aim to improve crops grown in the field (Rosenthal et al., 2011; Poorter et al.,
93
2016). Light is one of the most dynamic environmental factors that directly impacts on plant
94
performance, and it is therefore important to understand how plants acclimate to fluctuating light
95
environments such as those experienced under field conditions.
96
Plant acclimation to changes in irradiance can be categorised as (i) dynamic acclimation
97
which refers to a reversible biological process present within a given period of time (Walters and
98
Horton, 1994; Mullineaux, 2006; Okegawa et al., 2007; Athanasiou et al., 2010; Yin and Johnson,
99
2000; Tikkanen et al., 2010; Alter et al., 2012; Suorsa et al., 2012; Yamori, 2016); or (ii)
100
developmental acclimation which is defined as changes in morphology (e.g. leaf thickness and
101
density) resulting from a given growth light environment, and are largely irreversible (Weston et al.,
102
2000; Murchie, 2005), and is the focus of this study. The ability of plants to developmentally
103
acclimate to a given light environment is particularly well-demonstrated in leaves grown in sun and
104
shade conditions, which differ in photosynthetic efficiency, biochemistry (e.g. Rubisco content and
105
change in Photosystem II and I ratio), anatomy (e.g. chloroplast size and distribution) and
106
morphology (e.g. leaf mass area and thickness) (Givnish, 1988; Walters and Horton, 1994; Weston et
4 13, 2017 - Published by www.plantphysiol.org Downloaded from www.plantphysiol.org on February Copyright © 2017 American Society of Plant Biologists. All rights reserved.
107
al., 2000; Bailey et al., 2001; Bailey et al., 2004). Plants grown under high light intensity tend to
108
develop thicker leaves than those grown under low light intensity (Evans and Poorter, 2001), which
109
generally increases photosynthetic capacity per unit area improving the plant’s ability to utilize light
110
for carbon fixation (Terashima et al., 2006). Increased photosynthetic capacity is often strongly
111
correlated with the concentration of photosynthetic enzymes such as Rubisco, cytochrome f, H+-
112
ATPase and reaction centres (Foyer et al., 2012). Leaves acclimated to shade tend to have higher net
113
photosynthetic rates at lower light levels and a lower light compensation point compared to sun
114
leaves (Givnish 1988). To compare plants/species with different leaf thicknesses, previous studies
115
have used mass integrated photosynthesis as a proxy to assess the photosynthetic efficiency of
116
plants with different volumes of photosynthetic tissues (Garnier et al., 1999; Evans and Poorter,
117
2001; Wright et al., 2001). Previous studies investigating developmental acclimation have primarily
118
focused on the effect of light intensity, with less emphasis given to the effect of dynamic light during
119
growth, like that experienced under a natural environment. Fluctuations in light could have a
120
significant impact on acclimation processes during growth, and need to be investigated alongside
121
light intensity to assess the interaction between light regime and intensity.
122
Acclimation is the result of a balance between the cost of increasing leaf photosynthetic
123
capacity, which can be underutilized (Terashima et al., 2006; Oguchi et al., 2008), and the risk of
124
photo-oxidative damage if the mechanisms to dissipate excess energy received by the plant are not
125
sufficient (Li et al., 2009). Under natural environmental conditions, the random duration and
126
intensity of fluctuating light from passing clouds or leaf movements (sun- and shade-flecks) result in
127
incident light intensities below light saturation that reduce photosynthetic rates, whilst those
128
intensities greater than saturated lead to excess excitation energy that can result in short potential
129
“stress” periods and long term damage to leaf photosynthesis (Baker, 2008). Plants therefore
130
employ mechanisms that enable them to deal with these changes in excitation pressure, including
131
thermal dissipation of excitation energy. Such processes are termed non-photochemical quenching
132
(NPQ) and are mainly associated with changes in the xanthophyll cycle (Demmig-Adams and Adams,
133
1992; Muller et al., 2001) and protonation of PSII antenna proteins (Li et al., 2000; 2004) both of
134
which are linked to the proton gradient across the thylakoid membrane. Large diversity in light
135
acclimation exists between individuals and species (Murchie and Horton, 1997), partly due to the
136
random nature of light fluctuations and species-specific responses.
137
To date the majority of studies examining acclimation to fluctuating light conditions have
138
been carried out on plants grown under constant intensities of light and swapped to a simple light
139
pattern (consisting of one or more step changes in light intensity of different frequencies) (Yin and
140
Johnson, 2000; Tikkanen et al., 2010; Alter et al., 2012; Suorsa et al., 2012; Yamori, 2016). Under
5 13, 2017 - Published by www.plantphysiol.org Downloaded from www.plantphysiol.org on February Copyright © 2017 American Society of Plant Biologists. All rights reserved.
141
these light conditions, acclimation responses have often been monitored over a period of several
142
days (e.g. Alter et al., 2012; Athanasiou et al., 2010). Whilst this approach is powerful for studies on
143
the mechanisms of dynamic light acclimation, it fails to recognise the importance of how plants
144
developmentally acclimate to growth under fluctuating light intensities (Huxley, 1969), such as those
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found in the natural field environment (Frechilla et al., 2004). There are only a handful of studies
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that have examined the impact of “real” dynamic light environments on plant growth and
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performance, and as far as we are aware none of these have used a controlled environment to
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examine the direct impact of light. For example, Yamori (2016) revealed the importance of
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unpredictable variations in environmental growth conditions (including light) that led to a reduction
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in photosynthesis because plants were unable to fully acclimate to the highly dynamic variation in
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light. However in this study, plants were grown under natural environmental conditions that
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resulted in fluctuations in a number of environmental variables and therefore the impact of light
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alone on the acclimation response could not be distinguished. Kulheim et al. (2002) compared field
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grown non-photochemical quenching Arabidopsis mutants with those grown in controlled
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environment chambers under constant or variable light intensity, and demonstrated that NPQ is
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important for plant fitness in the field and under fluctuating environments reproduced in growth
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chambers. However, when the plants were grown under constant light conditions no effect on plant
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performance was observed, emphasizing the influence of growth environment on plant fitness.
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Although the study by Kulheim et al. (2002) was one of the first to examine the influence of dynamic
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and square wave growth light regimes on plant performance and growth, the dynamic light regime
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used in the controlled environment did not mimic that observed in the field.
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In order to fully understand how plants integrate fluctuations in incident light, and how this
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influences acclimation and modifies plant growth, there is a need to grow plants in a controlled but
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dynamic environment that mimics a light regime that would be experienced in the field. How plants
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perform under these conditions and the differences in responses with those grown in square wave
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light regimes is important, as models of steady state photosynthesis tend to overestimate
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photosynthesis under fluctuating light regimes (Naumburg and Ellsworth, 2002). The importance of
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developmental acclimation for plant performance has been demonstrated in “sun” (high light) and
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“shade” (low light) leaves. However, it is not known if developmental acclimation to fluctuating light
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intensity exists and if so how it may influence plant performance under dynamic light conditions,
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such as those experienced in a natural environment, and force us to rethink experimental growth
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conditions to draw conclusions on how plants will perform in the field. To address this, Arabidopsis
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plants were grown and measured under fluctuating and non-fluctuating (or square wave) light
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regimes at two different average intensities (high and low) (Fig. 1) and the performance of these
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plants and their ability to convert light energy into biomass evaluated.
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Results
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Photo-acclimation of plants grown under different light regimes
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Light response curves in which net CO2 assimilation rate (A) was measured as a function of
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photosynthetic photon flux density (PPFD) (Q) (A/Q curves, Fig. 2A) displayed similar A values at
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PPFDs below 250 µmol m-2 s-1 in plants grown under all the different light regimes: square wave high
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(SQH) and low (SQL) and fluctuating high (FLH) and low (FLL) light intensity. Measurements of A at
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PPFD above this value and light saturated assimilation rate (Asat, Table 1), were significantly greater
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in plants grown under high light intensity compared with those grown under low light,
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independently of the light regime (Fig.2A).
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Generally, photosynthesis is measured per unit leaf area, however this area also represents a
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volume of photosynthetic tissues that can differ among plants (e.g. different leaf thickness). To take
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into consideration photosynthesis per unit leaf volume, we integrated A by mass of dry leaf (Amass).
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There was significantly greater Amass in plants grown under fluctuating light regimes compared to
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those grown under square wave light regimes (Fig. 2B), and as expected a tendency for plants grown
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under high light regimes to have greater rates of Amass compared to plants grown under low light
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regimes.
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Dark respiration, derived from the A/Q curve (Rd-model) was significantly higher in plants grown under
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SQH and there was a general tendency for higher respiration in plants grown in square wave light
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intensity regimes compared with fluctuating regimes (Table. 1) in Rd-model as well as those measured
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during diurnals (Rd-diurnal). However it should be noted that, Rd-diurnal measured at the start of the
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diurnal was lower than that Rd-model determined from the A/Q analysis. Plants grown under SQH also
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had a significantly higher light compensation point (Γ) compared to plants grown under fluctuating
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light regimes (Table 1).
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The large differences observed in the response of A to PPFD between plants grown under low and
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high light intensity, was less significant for Photosystem II (PSII) operating efficiency (Fq’/Fm’) (Fig.
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2C). Response of Fq’/Fm’ to PPFD was mainly driven by changes in the PSII efficiency factor (Fq’/Fv’)
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(Fig. 2D). Fq’/Fm’ was also affected, although to a lower extent, by the maximum efficiency of PSII
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(Fv’/Fm’) which was higher in plants grown under high PPFD (Fig. 2E), with low values illustrating
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greater non-photochemical quenching. In general plants grown under fluctuating regimes had a
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higher Fv’/Fm’ compared with those grown under square wave, particularly when measured under
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high PPFDs. Plants grown under SQL showed the lowest values in both quenching parameters: Fq’/Fv’
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and Fv’/Fm’. Non-photochemical quenching (NPQ) increased more rapidly at low light intensity in
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plants grown under SQL compared to plants grown in the other lighting regimes, and in general NPQ
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had a tendency to be lower in plants grown under fluctuating light (Fig. 2F).
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Leaf properties in plants acclimated to different light regimes
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Leaf absorbance (measured after 28 days of growth) was significantly different between plants
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grown in the different light regimes (P
