Download The Regulatory Properties of Rubisco Activase

The Regulatory Properties of Rubisco Activase Differ
among Species and Affect Photosynthetic Induction
during Light Transitions1[W][OA]
A. Elizabete Carmo-Silva 2 and Michael E. Salvucci*
United States Department of Agriculture, Agricultural Research Service, Arid-Land Agricultural Research
Center, Maricopa, Arizona 85138
Rubisco’s catalytic chaperone, Rubisco activase (Rca), uses the energy from ATP hydrolysis to restore catalytic competence to
Rubisco. In Arabidopsis (Arabidopsis thaliana), inhibition of Rca activity by ADP is fine tuned by redox regulation of the
a-isoform. To elucidate the mechanism for Rca regulation in species containing only the redox-insensitive b-isoform, the
response of activity to ADP was characterized for different Rca forms. When assayed in leaf extracts, Rubisco activation was
significantly inhibited by physiological ratios of ADP to ATP in species containing both a-Rca and b-Rca (Arabidopsis and
camelina [Camelina sativa]) or just the b-Rca (tobacco [Nicotiana tabacum]). However, Rca activity was insensitive to ADP
inhibition in an Arabidopsis transformant, rwt43, which expresses only Arabidopsis b-Rca, although not in a transformant of
Arabidopsis that expresses a tobacco-like b-Rca. ATP hydrolysis by recombinant Arabidopsis b-Rca was much less sensitive to
inhibition by ADP than recombinant tobacco b-Rca. Mutation of 17 amino acids in the tobacco b-Rca to the corresponding
Arabidopsis residues reduced ADP sensitivity. In planta, Rubisco deactivated at low irradiance except in the Arabidopsis rwt43
transformant containing an ADP-insensitive Rca. Induction of CO2 assimilation after transition from low to high irradiance was
much more rapid in the rwt43 transformant compared with plants containing ADP-sensitive Rca forms. The faster rate of
photosynthetic induction and a greater enhancement of growth under a fluctuating light regime by the rwt43 transformant
compared with wild-type Arabidopsis suggests that manipulation of Rca regulation might provide a strategy for enhancing
photosynthetic performance in certain variable light environments.
The activity of Rubisco, the enzyme that catalyzes
CO2 assimilation in photosynthesis, is regulated by
Rubisco activase (Rca), a specific catalytic chaperone
(Spreitzer and Salvucci, 2002; Portis, 2003). Like other
AAA+ ATPases (Snider et al., 2008), Rca uses the energy from ATP hydrolysis to remodel the conformation of its target protein, Rubisco. The conformational
changes induced by Rca restore catalytic competence
to Rubisco active sites that have been inactivated by
the unproductive binding of sugar phosphates, including the substrate ribulose 1,5-bisphosphate (RuBP;
Wang and Portis, 1992). Because of the requirement for
ATP hydrolysis and the inhibition of activity by ADP
(Robinson and Portis, 1988, 1989), Rca adjusts the rate
1
This work was supported by the Division of Chemical Sciences,
Geosciences, and Biosciences, Office of Basic Energy Sciences, U.S.
Department of Energy (Photosynthetic Systems grant no. DE–AI02–
97ER20268 to M.E.S.).
2
Present address: Plant Biology and Crop Science, Rothamsted
Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Michael E. Salvucci ([email protected]).
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.112.213348
of CO2 fixation to the rates of electron transport activity via changes in the activation state of Rubisco
(Salvucci et al., 1985). As a result of this coordinate
regulation, the light response of Rubisco activation
closely resembles the light response of CO2 assimilation, and the levels of RuBP under steady-state conditions are relatively constant over a wide range of
irradiance levels (Perchorowicz et al., 1981; Dietz and
Heber, 1984).
Many plant species express two isoforms of Rca, a
and b, that are both active in ATP hydrolysis and
Rubisco activation (Shen et al., 1991; Salvucci et al.,
2003). In some plant species, these isoforms are the
products of an alternative splicing event that generates
two polypeptides, which are identical except for a 20to 30-amino acid extension at the C terminus of the
longer a-isoform (Werneke et al., 1989). In cotton
(Gossypium hirsutum), soybean (Glycine max), and presumably other plant species, separate genes encode the
two isoforms of Rca (Salvucci et al., 2003; Yin et al.,
2010). In these species, the amino acid sequences of the
overlapping regions of the a- and b-polypeptides are
very similar, and the C-terminal extension of the longer a-isoform is similar to the extension produced by
alternative splicing (Supplemental Fig. S1).
Our current understanding of the role of the two
Rca isoforms is based primarily on investigations
with Arabidopsis (Arabidopsis thaliana; Zhang and
Portis, 1999; Zhang et al., 2001; Wang and Portis,
2006). The C-terminal extension of the a-Rca contains
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Carmo-Silva and Salvucci
two redox-regulated Cys residues that are modulated
by thioredoxin f (Zhang and Portis, 1999). When these
residues are oxidized to a disulfide, the affinity for
ATP decreases and enzyme activity is more sensitive
to inhibition by ADP. Physiological ratios of ADP to
ATP significantly inhibit the activity of the Arabidopsis a-Rca when in the oxidized state, but inhibition
is much less when this isoform has been reduced by
thioredoxin. In contrast, the shorter Arabidopsis b-Rca
is not redox regulated and is less sensitive to inhibition
by ADP (Zhang and Portis, 1999). Mixing experiments
with recombinant Rca have shown that the properties
of a-Rca are conferred to the heterooligomer, providing a mechanism for redox regulating the Rca holoenzyme (Zhang et al., 2001). In this way, Rca is similar
to the chloroplastic glyceraldehyde 3-P dehydrogenase
(GAPDH), which also has both redox-regulated (GAPB) and non-redox-regulated (GAP-A) forms that differ
by a C-terminal extension (Baalmann et al., 1996). Like
Rca (Zhang and Portis, 1999; Zhang et al., 2001), redox
regulation of two Cys residues in the extension exerts
master control over the mixed GAPDH oligomer.
Some plant species, including members of the Solanaceae family, as well as maize (Zea mays) and green
algae, express only the shorter b-Rca (Salvucci et al.,
1987). The b-Rca in these species is not responsive to
redox regulation, even though the activation state of
Rubisco in these plants is modulated by irradiance
(Salvucci and Anderson, 1987) and seems to be associated with the redox status of the chloroplast (Ruuska
et al., 2000). With GAPDH, all higher plants appear to
have both chloroplastic isoforms, but the non-redoxsensitive form, Gap-A, can be regulated indirectly by
thioredoxin through the binding of the small chloroplast protein CP12 (Trost et al., 2006). By analogy, a
similar association with CP12 or a CP12-like protein
could provide a means of conferring redox sensitivity
to b-Rca in species that have only this Rca isoform.
However, no association of Rca was observed when
the native CP12 complex and other high-molecularmass species were isolated from tobacco (Nicotiana
tabacum) chloroplasts (Carmo-Silva et al., 2011b).
In this study, the regulation of b-Rca activity was
examined both in vivo and in vitro in plant species that
contain only one (i.e. b-) or both (a- and b-) Rca isoforms. The response of enzyme activity to physiological ratios of ADP to ATP was measured for the native
Rca in leaf extracts, as well as for recombinant Arabidopsis and tobacco enzymes, to determine the sensitivity of b-Rca to ADP in different species. In addition,
experiments were conducted with transgenic Arabidopsis plants containing variants of b-Rca to determine
the link between Rca regulation and photosynthetic
induction. The results suggest a new strategy for enhancing photosynthetic performance under variable
light environments based on altering the regulatory
properties of Rca to increase the rate of photosynthetic
induction.
RESULTS
ADP Sensitivity of Rubisco Activation by Rca in
Leaf Extracts
The sensitivity of Rca activity to inhibition by ADP
was determined by measuring the rate of Rubisco activation in leaf extracts in the presence of physiological
ratios of ADP to ATP (Fig. 1). The rates of Rubisco
activation were inhibited by increasing ratios of ADP
to ATP in Arabidopsis and camelina (Camelina sativa),
two species that express both the a-Rca and b-Rca
isoforms, and in tobacco, a species that expresses only
Figure 1. Response of Rca activity to ADP in plants with different Rca forms. The rate of Rubisco activation by Rca in leaf
extracts was measured at 30˚C in the presence of either 5 mM ATP plus an ATP-regenerating system (0.00 ADP/ATP) or a ratio of
0.11 or 0.33 ADP/ATP (total nucleotide concentration of 5 mM). The Arabidopsis transgenic line rwt43 expresses only the
Arabidopsis b-Rca and the line Tob-DAt(S2) expresses a chimeric b-Rca that resembles the tobacco protein but has the Rubisco
recognition domain from Arabidopsis. Rates of Rubisco activation are expressed as vi/vc (the inhibited velocity/control velocity,
i.e. the rates determined in the presence of ADP relative to the control rate with ATP alone). Control rates were 0.17 6 0.02,
0.20 6 0.00, 0.04 6 0.01, 0.10 6 0.01, and 0.13 6 0.01 Rubisco sites activated per min for Arabidopsis wild type, rwt43, TobDAt(S2), tobacco, and camelina, respectively. Values are means 6 SE of four to six samples. Different letters at the base of each
bar denote significant differences (P , 0.05) between the rates at each ratio of ADP to ATP.
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Rubisco Activase Regulation
b-Rca. A similar response was observed in the Arabidopsis line Tob-DAt(S2), a transformant of the rca null
mutant that expresses a chimeric b-Rca, which is
identical to tobacco Rca except for inclusion of the
Rubisco recognition domain from Arabidopsis (Kumar
et al., 2009). Compared with the control rate measured
in the absence of ADP, the rates of Rubisco activation
in these plants were inhibited by 40% to 60% at 0.11
ADP/ATP and by 70% to 80% at 0.33 ADP/ATP (Fig.
1). In contrast, the activity of Rca in leaf extracts of the
Arabidopsis line rwt43, a transformant of the rca null
mutant that expresses only the Arabidopsis b-Rca
(Zhang et al., 2002), was not inhibited by these ratios of
ADP to ATP (P . 0.05).
tobacco (Servaites et al., 1986), but not in Arabidopsis
and other plant species (Salvucci et al., 1986).
In species that do not accumulate 2-carboxyarabinitol1-P, the activation state of Rubisco in the dark, when the
RuBP concentration is low, is dependent on the concentration of CO2 and can be greater than at low irradiance
(Perchorowicz et al., 1981; Salvucci et al., 1986). In the
dark, Rubisco sites are free of RuBP and readily carbamylate with the available CO2. In the light, the production of RuBP can generate a dead-end complex, when it
binds to uncarbamylated Rubisco. Thus, in the light,
the activation state of Rubisco is determined not by the
concentration of CO2, except at very low CO2, but by the
balance between inactivation by RuBP and reactivation
by Rca (Salvucci et al., 1985, 1986). To achieve a constant
low level of Rubisco activation in both wild-type Arabidopsis and the rwt43 transformant, plants were placed in
the dark (i.e. a condition where the activity of Rca does
not affect Rubisco activation). After 2 h in the dark at
ambient CO2, the activation state of Rubisco in both
wild-type Arabidopsis and the rwt43 transformant was
about 50% (Supplemental Table S1). When plants were
transferred from dark to low irradiance, the activation
state of Rubisco decreased in wild-type plants but increased in the rwt43 transformant. Thus, the Rca in the
rwt43 transformant produced a high Rubisco activation
state at low irradiance, both upon transition from high to
low irradiance (Table I) and from dark to low irradiance
(Supplemental Table S2).
At high irradiance, the amount of the Rubisco substrate, RuBP, was slightly lower in the Arabidopsis rwt43
transformant than in wild-type plants (Supplemental
Table S2). At low irradiance, RuBP levels in the rwt43
transformant were less than half the levels in wild-type
plants (Supplemental Table S2; Zhang et al., 2002),
consistent with the much higher activation state of
Rubisco in the transformant (Table I). In contrast, the
amounts of Fru-1,6-bisP and triose phosphate in the
Modulation of Rubisco Activation by Irradiance in Plants
with Different Rca Forms
To relate differences in ADP sensitivity to the in
planta activity of Rca, the activation state of Rubisco
was measured in plants exposed to high irradiance
and after transfer to low irradiance (Table I). The activation state of Rubisco decreased when plants were
transferred from high to low irradiance in wild-type
Arabidopsis, tobacco, and camelina and in the Arabidopsis Tob-DAt(S2) transformant. In contrast, there was
no significant difference (P . 0.05) in Rubisco activation state at high and low irradiance in the Arabidopsis
rwt43 transformant. Lower initial Rubisco activities
accompanied the reduced Rubisco activation states at
low irradiance, whereas Rubisco total activities were
not significantly affected (P . 0.05) by irradiance level,
except in tobacco (Table I). In all likelihood, the decrease in total Rubisco activity in tobacco was caused
by 2-carboxyarabinitol-1-P, a naturally occurring tightbinding inhibitor of Rubisco that accumulates in the
dark and at low light in many plant species, including
Table I. Effect of irradiance on the activation state of Rubisco in plants containing different Rca forms
Leaf discs were illuminated at 1,000 or 1,800 mmol m22 s21 for 30 min (high irradiance) followed by
30 mmol m22 s21 for 60 min (low irradiance) and then immediately frozen in liquid nitrogen. Frozen leaf
discs were extracted and immediately assayed for initial and total Rubisco activity. The activation state was
calculated as the initial activity divided by the total activity at high irradiance multiplied by 100. Values
are means 6 SE (n as indicated). Different letters after the values denote significant difference between the
values at low and high irradiance (P , 0.05).
Plant
Irradiance
mmol m
Arabidopsis wild type
Arabidopsis rwt43
Arabidopsis Tob-DAt(S2)
Tobacco
Camelina
22
1,000
30
1,000
30
1,000
30
1,800
30
1,800
30
n
Rubisco
Initial Activity
21
mmol min
s
6
5
6
6
5
4
4
4
4
3
0.79
0.46
1.03
1.01
1.05
0.46
0.82
0.27
0.79
0.35
6
6
6
6
6
6
6
6
6
6
Total Activity
21
0.07 a
0.10 b
0.16 a
0.14 a
0.14 a
0.21 b
0.12 a
0.04 b
0.04 a
0.04 b
mg
21
Activation State
protein
0.87
0.83
1.12
1.10
1.28
1.17
0.87
0.47
0.75
0.70
6
6
6
6
6
6
6
6
6
6
0.07 a
0.10 a
0.16 a
0.15 a
0.16 a
0.28 a
0.11 a
0.03 b
0.04 a
0.04 a
Plant Physiol. Vol. 161, 2013
%
91 6 8 a
53 6 12 b
92 6 14 a
90 6 12 a
81 6 11 a
36 6 16 b
93 6 14 a
31 6 4 b
105 6 5 a
46 6 6 b
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Carmo-Silva and Salvucci
rwt43 transformant were similar to the amounts in
wild-type plants at both high and low irradiance.
ADP Inhibition of Recombinant b-Rca ATPase Activity
To determine if the difference in ADP sensitivity was
an intrinsic characteristic of the enzyme, the effect of
various ADP/ATP ratios on the ATPase activity of
recombinant b-Rca from Arabidopsis and tobacco was
compared (Fig. 2). At 0.11 ADP/ATP, the activity of
the Arabidopsis b-Rca was not significantly different
(P . 0.05) from the activity measured in the presence
of ATP alone, whereas the activity of the tobacco enzyme was inhibited by about 50%. At 0.33 ADP/ATP,
the activity of the Arabidopsis enzyme was inhibited
by about 40% compared with 87% for tobacco b-Rca.
Thus, the b-Rca from the two species differed markedly in their sensitivity to inhibition by ADP. These
differences were consistent with the different effects of
ADP on Rubisco activation activity measured in leaf
extracts (Fig. 1) as well as the different response of
Rubisco activation to low irradiance measured in planta
(Table I).
Comparison of the amino acid sequences of the
b-Rca from Arabidopsis and tobacco showed that the
two proteins are 82% identical (Fig. 3). While differences between the two polypeptides are distributed
throughout the protein, residues affecting nucleotide
binding are likely to fall within the AAA+ motif
(Henderson et al., 2011; Stotz et al., 2011). Also, it is
likely that ADP sensitivity is conferred by a residue(s)
outside the Rubisco recognition domain, since Rca
activity in leaf extracts of the Arabidopsis Tob-DAt(S2)
transformant was inhibited by physiological ratios of
ADP to ATP (Fig. 1).
Differences in the Rca sequence between tobacco
and Arabidopsis include two nonconservative residues
in the AAA+ module near the nucleotide-binding
domain: residue 125 (tobacco numbering) near the
Walker A motif and residue 166 near the Walker B
motif. These residues were mutated separately in the
tobacco b-Rca to the corresponding residue in Arabidopsis. Inhibition of the ATPase activity of the recombinant R125A and N166K proteins by ADP was
similar to the wild-type tobacco b-Rca (Fig. 2). Consequently, multiple mutations were introduced in the
tobacco b-Rca, changing these two plus 15 other residues to the corresponding residues in Arabidopsis.
These changes were targeted to the AAA+ module but
outside the Rubisco recognition domain (Fig. 3). No
changes were made in the N-terminal domain because previous studies have shown that removal of
the first 50 residues of the tobacco Rca does not affect
ATP binding or hydrolysis (van de Loo and Salvucci,
1996; Stotz et al., 2011). The mutated enzyme, designated Tob-DAt(n=17), catalyzed ATP hydrolysis at rates
equivalent to wild-type tobacco b-Rca but was much
less sensitive to inhibition by ADP, more closely
resembling the Arabidopsis than the tobacco b-Rca
(Fig. 2).
Light Induction of Photosynthesis in Plants with Different
Rca Forms
The rate of photosynthetic induction (i.e. the time
required to reach a constant steady-state rate of net
CO2 assimilation upon transition from low to high irradiance) has been attributed to the rate of Rubisco
activation by Rca (Woodrow et al., 1996; Mott and
Woodrow, 2000). After 1 h at low irradiance, photosynthetic induction upon transition to high irradiance
took as long as 30 min in wild-type Arabidopsis,
camelina (data not shown), tobacco, and the Arabidopsis Tob-DAt(S2) transformant (Fig. 4). In tobacco,
Figure 2. Effect of ADP on the ATPase activity of recombinant Arabidopsis and tobacco b-Rca as well as two point mutants and
a 17-residue substitution mutant of tobacco Rca. The rate of ATP hydrolysis was determined in the presence of 5 mM adenine
nucleotide at the indicated ratios of ADP to ATP. Rates of ATP hydrolysis are expressed as vi/vc (the inhibited velocity/control
velocity, i.e. the rates determined in the presence of ADP relative to the control rate with ATP alone). Control rates were 0.84 6
0.06, 0.52 6 0.01, 0.45 6 0.03, 0.68 6 0.03, and 0.55 6 0.02 units mg21 protein for Arabidopsis, tobacco, R125A, N166K,
and the 17-residue substitution mutant Tob-DAt(n=17), respectively. Values are means 6 SE of three to five measurements.
Different letters at the base of each bar denote significant differences (P , 0.05) between the rates at each ratio of ADP to ATP.
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Rubisco Activase Regulation
Figure 3. Multiple sequence alignment
of b-Rca from tobacco, Arabidopsis,
and two tobacco-Arabidopsis chimeras, Tob-DAt(S2) and Tob-DAt(n=17).
Residues common to all sequences are
in blue. Tobacco residues that are different or missing in the Arabidopsis
sequence are in red boldface. Dashed
lines immediately above the tobacco
sequence indicate the positions of the
N terminus and Rubisco recognition
domain (dark red). Asterisks indicate the
tobacco residues Arg-125 and Asn-166
that were changed individually to the
corresponding residues in Arabidopsis.
Residues in the tobacco sequence that
were changed to the corresponding
Arabidopsis residues to make the TobDAt(n=17) mutant protein are underlined.
photosynthetic induction was particularly slow, probably
because of the accumulation of 2-carboxyarabinitol-1-P
(Table I; Hammond et al., 1998). The rate of photosynthetic induction in these plants increased with decreasing
time of exposure to low irradiance.
In contrast to the results with plants containing
ADP-sensitive Rca forms, the rate of photosynthetic
induction in the Arabidopsis rwt43 transformant was
almost instantaneous and was unaffected by the time
of exposure at low irradiance. Measurement of chlorophyll fluorescence following an increase from low to
moderate (i.e. 200 mmol m22 s21) irradiance showed that
nonphotochemical quenching (NPQ) exhibited a transient increase during photosynthetic induction in wildtype Arabidopsis but not in the rwt43 transformant
(Fig. 5).
Relaxation times, determined as the reciprocal of the
apparent rate constant during the initial phase of
photosynthetic induction (Woodrow and Mott, 1989),
were measured in wild-type Arabidopsis and the
rwt43 transformant to quantify the rates of photosynthetic induction (Fig. 6). Measurements were conducted at 15°C, 23°C, and 35°C to determine if the
regulation of Rca affects photosynthetic induction at
suboptimal, optimal, and supraoptimal temperatures
(Yamori et al., 2012). Compared with wild-type Arabidopsis, the rate of photosynthetic induction was
twice as fast in the rwt43 transformant at 15°C and
three times faster at 23°C and 35°C. For both wild-type
Arabidopsis and the rwt43 transformant, the rate of
photosynthetic induction was fastest at 23°C. This
temperature coincides with the temperature optimum
of CO2 assimilation (Kim and Portis, 2005) as well as
the optimum for Rca activity in Arabidopsis (CarmoSilva and Salvucci, 2011). The longer relaxation time at
35°C compared with 23°C in wild-type plants was
consistent with inhibition of Rca activity at the higher
temperature.
Growth of Plants with Different Rca Forms under Variable
Light Environments
Measurements of biomass accumulation indicated
that the Arabidopsis rwt43 transformant plants grew
more slowly than the wild type in air under control
conditions of light and temperature. After 5 weeks,
the average dry weight of the wild type and the
rwt43 transformant were 95.5 6 4.1 and 67.7 6 3.0
mg, respectively. Even though the rates of photosynthetic induction differed markedly, the light
response of photosynthesis was very similar for wildtype Arabidopsis and the rwt43 transformant plants
(Supplemental Fig. S2). At saturating irradiance, the
CO2 assimilation rates were higher for the wild-type
plants compared with the rwt43 plants, 11.5 and 9.5
mmol m22 s21, respectively. At the irradiance used for
growth, the CO2 assimilation rates were 2.4 6 0.1 and
1.9 6 0.1 mmol m22 s21 for the wild type and the
rwt43 transformant, respectively. Rca expression in
the rwt43 transformant line is about 70% of the level
present in wild-type Arabidopsis (Zhang et al., 2002)
but certainly not limiting for Rubisco activation
(Table I).
Plant Physiol. Vol. 161, 2013
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Carmo-Silva and Salvucci
Figure 4. Induction of CO2 assimilation upon the transition from low to high irradiance in plants with different Rca forms.
Plants were placed at high irradiance: 1,000 mmol m22 s21 for Arabidopsis or 1,800 mmol m22 s21 for tobacco. After 20 min,
the irradiance was decreased to 30 mmol m22 s21 for 5, 15, 30, and 60 min. The irradiance was then increased to 1,000 mmol
m22 s21 for Arabidopsis or 1,800 mmol m22 s21 for tobacco (time zero), and the rate of net CO2 assimilation was measured
continuously thereafter. Measurements were conducted at 23˚C for Arabidopsis or at 28˚C for tobacco. Assimilation rates for
tobacco were corrected for differences in stomatal conductance by adjusting the rates to an intercellular CO2 concentration of
280 mmol mol21. No correction was required for Arabidopsis. The relative photosynthetic rate represents the fraction of the
maximum rate, measured after 20 min at high irradiance: 9.7 6 0.2, 8.0 6 0.2, 6.6 6 0.1, and 23.4 6 0.9 mmol m22 s21 for
Arabidopsis wild type, the rwt43 and Tob-DAt(S2) transformants, and tobacco, respectively. Values are means 6 SE of measurements taken with four to five separate plants.
On an area basis, soluble protein was higher in the
rwt43 plants than in wild-type plants, 0.28 6 0.01 and
0.25 6 0.02 mg cm22, respectively, while chlorophyll
content was lower, 0.027 6 0.001 and 0.031 6 0.001 mg
cm22, respectively. When adjusted for differences in
soluble protein, Rubisco total activity was higher on an
area basis in the rwt43 plants compared with the wild
type (Table I). Kim and Portis (2005) reported that the
response of CO2 assimilation rate to intercellular CO2
concentration was similar in the rwt43 and wild-type
plants, and we confirmed this result in separate experiments (data not shown). The maximal velocity of
carboxylation calculated from our measurements of
the response of the rate of CO2 assimilation to the intercellular CO2 concentration was about 42 mmol m22
s21 in both wild-type and rwt43 plants, indicating that
Rubisco content was similar in the wild type and the
transformant. In addition, Kim and Portis (2005)
reported that the temperature response of CO2 assimilation rate was nearly identical in the rwt43 and wildtype plants.
Arabidopsis wild-type and rwt43 transformant
plants were grown under several other conditions
to determine if the high activation state of Rubisco
at low irradiance and its effect on photosynthetic
induction, RuBP levels, and NPQ were detrimental
to growth. Compared with plants grown under
control conditions (i.e. 10-h photoperiod at 175
mmol m22 s21), both Arabidopsis wild type and the
rwt43 transformant exhibited an increase in shoot
dry weight when grown at twice the irradiance
(Supplemental Fig. S3). Growth under an alternating irradiance regime (i.e. 420 and 20 mmol m22 s21 at
1-h intervals for 15 h) had no adverse effect on
the growth of either wild-type Arabidopsis or the
rwt43 transformant. In fact, the rwt43 plants
responded more positively to growth under a fluctuating light condition than the wild type. In contrast, biomass accumulation decreased for both the
wild-type and the rwt43 when darkness was used
instead of low irradiance for the alternating light
condition.
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Rubisco Activase Regulation
b-Rca when in association with the respective a-Rca.
Rca activity was also inhibited by ADP in the Arabidopsis Tob-DAt(S2) transformant, containing a chimeric
b-Rca identical to the tobacco enzyme but with the
Rubisco recognition domain of Arabidopsis. Mutation
Figure 5. Time course of NPQ upon transition from low to moderate
irradiance in wild-type Arabidopsis and the rwt43 transformant. Plants
were initially dark adapted for 60 min to determine the maximal
chlorophyll fluorescence in the dark-adapted leaves (Fm). Plants were
then placed at 1,000 mmol m22 s21 for 20 min to fully activate
Rubisco, and the irradiance was then decreased to 30 mmol m22 s21.
After 60 min (time zero), the irradiance was increased to 200 mmol m22
s21 and the maximal chlorophyll fluorescence in the light (Fm9) was
measured continuously for 15 min at 23˚C. NPQ was calculated as
(Fm 2 Fm9)/Fm9. Values are means 6 SE of measurements taken with four
separate plants.
DISCUSSION
Species Differences in the Regulatory Properties of Rca
The response of Rca activity to physiological ratios
of ADP to ATP showed that the regulatory properties
of b-Rca in Arabidopsis differed from those of b-Rca in
tobacco. In wild-type Arabidopsis, b-Rca is paired
with a-Rca, and the interaction of the two isoforms
affects the sensitivity of the holoenzyme to inhibition
by ADP (Zhang et al., 2001; Wang and Portis, 2006).
That the Cys residues in the C-terminal extension are
conserved in a-Rca from diverse species, including
those produced from separate genes (Supplemental
Fig. S1), suggests that redox regulation is necessary for
the proper functioning of the a-Rca plus b-Rca holoenzyme, at least under some conditions. In spinach
(Spinacia oleracea) and Arabidopsis, the ratio of ADP to
ATP is thought to remain constant over a range of irradiances that affect Rubisco activation (Brooks et al.,
1988). Thus, in these species, redox sensitivity of a-Rca
would be essential for regulating Rubisco activation in
response to irradiance.
The b-isoform of Rca does not contain a redoxreactive Cys pair and is unaffected by changes in redox state mediated by thioredoxin f (Zhang and Portis,
1999). Thus, the mechanism for the regulation of Rca in
plant species like tobacco that express only the nonredox-regulated b-isoform has been enigmatic. b-Rca
from tobacco was acutely sensitive to inhibition
by ADP, resembling the response of the Arabidopsis
Figure 6. Induction of CO2 assimilation upon transition from low to
high irradiance in Arabidopsis wild-type and rwt43 transformant plants
at suboptimal (15˚C), optimal (23˚C), and supraoptimal (35˚C) temperatures. Plants were placed at 1,000 mmol m22 s21. After 20 min, the
irradiance was decreased to 30 mmol m22 s21 for 60 min. The irradiance
was then increased to 1,000 mmol m22 s21 (time zero), and the rate of
net CO2 assimilation was measured continuously thereafter. Values are
means 6 SE of measurements taken with four to five separate plants.
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Carmo-Silva and Salvucci
of 17 residues in the tobacco b-Rca to the corresponding residues in the Arabidopsis b-Rca resulted in
a b-Rca [i.e. Tob-DAt(n=17)] that was more similar to
Arabidopsis than tobacco b-Rca in its response to
physiological ratios of ADP to ATP. Therefore, one or
more of these 17 residues (Fig. 3) confer sensitivity to
inhibition by ADP to the tobacco b-Rca.
The b-Rca in the Arabidopsis Tob-DAt(S2) transformant is not redox sensitive and strongly resembles
the tobacco b-Rca, making it the equivalent of a tobacco b-Rca in an Arabidopsis background. In the
Arabidopsis Tob-DAt(S2) plants, Rubisco deactivated
under low irradiance, indicating that the ADP/ATP
ratio must have decreased to a level that was inhibitory for the chimeric b-Rca. In contrast, Rubisco did
not deactivate after transition from high to low irradiance and activated after transition from dark to low
irradiance in the Arabidopsis rwt43 transformant, indicating that the ADP/ATP ratio at low irradiance was
not inhibitory for the Arabidopsis b-Rca. Taken together, these results indicate that changes in the ADP/
ATP ratio play a much greater role in regulating Rca
activity than previously suggested (Brooks et al., 1988;
Zhang and Portis, 1999; Ruuska et al., 2000). Moreover,
these changes alone might explain the regulation of
Rubisco activation in plants like tobacco that contain
an ADP-sensitive b-Rca.
It is not clear why different mechanisms have
evolved for regulating Rca. Redox regulation of Rca
provides fine control over the sensitivity of the holoenzyme to inhibition by ADP (Zhang and Portis, 1999;
Zhang et al., 2001). However, this level of regulation
does not seem to be essential in all plant species.
ATPase activity of cotton and creosote (Larrea tridentata) b-Rca was inhibited by ADP (A.E. CarmoSilva and M.E. Salvucci, unpublished data), indicating
that ADP inhibition of b-Rca varies among plant species and even occurs in species that also express an
a-Rca. Similarly, tobacco expresses several different
b-Rca proteins (Qian and Rodermel, 1993) that could
vary in their sensitivities to inhibition by ADP. However, measurements of the recombinant versions of the
products of the two main tobacco Rca genes (i.e. RCA1
and RCA2) showed that their responses to ADP/ATP
were similar (A.E. Carmo-Silva and M.E. Salvucci,
unpublished data).
Certain plant species like Arabidopsis, camelina,
and spinach express equal amounts of a-Rca and
b-Rca, while others like rice (Oryza sativa) and wheat
(Triticum aestivum) accumulate much more b-Rca
than a-Rca (Salvucci et al., 1987; Fukayama et al.,
2012). In still others like maize, only b-Rca has been
detected at the protein level under nonstress conditions (Salvucci et al., 1987; Vargas-Suárez et al., 2004),
even though the genome contains an open reading
frame that encodes for an a-Rca (accession no.
NM_001175017.1). Evidence has been presented for the
expression of a high-molecular-mass Rca polypeptide,
possibly the a-isoform, in some maize cultivars during
heat stress (Sánchez de Jiménez et al., 1995; Ristic et al.,
2009), similar to the up-regulation of a-Rca expression
in rice (Wang et al., 2010). Detailed analysis of the kinetic and thermal properties of the various a-Rca and
b-Rca forms is necessary to determine how patterns of
Rca isoform expression might exert control over enzyme activity.
Light Induction of Photosynthesis and in Vivo Regulation
of Rubisco Activation
In previous studies, Mott, Woodrow, and colleagues
demonstrated that Rca determines the rate of induction of photosynthetic CO2 assimilation following an
increase in irradiance (Woodrow and Mott, 1989;
Woodrow et al., 1996; Mott and Woodrow, 2000).
Using transgenic tobacco and Arabidopsis plants with
reduced amounts of Rca, they showed that the apparent rate constant for the induction of net CO2 assimilation was determined by the rate of Rubisco
activation by Rca (Mott et al., 1997; Hammond et al.,
1998). Similar findings were recently reported for
transgenic rice plants with altered amounts of Rca
(Fukayama et al., 2012; Yamori et al., 2012). The relaxation times (i.e. the reciprocal of the apparent rate
constant for the induction of net CO2 assimilation;
Woodrow and Mott, 1989) were shorter in rice plants
that overexpressed the b-Rca from barley (Hordeum
vulgare) and maize (Fukayama et al., 2012), consistent
with the predictions of the model presented by Mott
and Woodrow (2000).
In this study, the ADP sensitivity of Rca was correlated with the light modulation of Rubisco activation
state and the light induction of photosynthesis. In the
Arabidopsis rwt43 transformant, containing only the
ADP-insensitive Arabidopsis b-Rca, Rubisco did not
deactivate at low irradiance. As a result, the induction
of CO2 assimilation was almost instantaneous, because
Rubisco was in a highly activated state even under low
irradiance and did not require a period of time to activate when the irradiance was increased.
Rubisco deactivated at low irradiance in tobacco as
well as in the Arabidopsis Tob-DAt(S2) transformant,
both of which contain a b-Rca that exhibits marked
inhibition by ADP. Deactivation of Rubisco at low irradiance was also observed in wild-type Arabidopsis
and in camelina, both of which contain a mixed a-Rca
and b-Rca holoenzyme that is sensitive to ADP/ATP
inhibition in a redox-dependent manner. Because
Rubisco deactivated under low irradiance in all of
these plants, induction of CO2 assimilation was much
slower upon the transition from low to high irradiance
than in the Arabidopsis rwt43 transformant. Also,
unlike the rwt43 plants, the rate of induction increased
in these plants with increasing time at low irradiance.
Thus, by controlling the activation state of Rubisco, the
regulatory properties of Rca, and specifically the sensitivity to inhibition by ADP determines the rate of
photosynthetic induction upon the transition from low
to high irradiance.
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Rubisco Activase Regulation
Although qualitatively similar, the effect of altered
Rca regulation on photosynthetic induction differs
mechanistically from the effects obtained by overexpressing Rca (Fukayama et al., 2012; Yamori et al.,
2012). In transgenic rice that overexpresses barley and
maize b-Rca, Rubisco still deactivated at low irradiance, but the higher concentration of Rca promoted
faster rates of Rubisco activation after the transition
to high irradiance (Yamori et al., 2012). The regulatory properties of rice a-Rca and b-Rca as well as
those of maize and barley b-Rca are unknown, but
the deactivation of Rubisco at low irradiance even in the
Rca-overexpressing lines indicates that Rca in the transgenic plants was inhibited by ADP. In contrast, Rubisco
in the Arabidopsis rwt43 transformant maintained a high
activation state even under low irradiance because of the
altered regulation of Rca. As a result, the rate of photosynthetic induction in the Arabidopsis rwt43 transformant was extremely rapid, because the rate was no longer
dependent on the rate of Rubisco activation by Rca.
The Functional Significance of Regulating Rca
The pioneering work of Perchorowicz et al. (1981)
showed that when Rubisco deactivates under low irradiance, RuBP levels generally exceed the concentration necessary to saturate Rubisco active sites. By
keeping RuBP levels above the binding site concentration, light activation of Rubisco could potentially provide a mechanism to maintain metabolic homeostasis
under changing irradiance levels and, thus, avoid lags
in the resumption of CO2 assimilation caused by a slow
rate of substrate regeneration through autocatalysis.
However, the data from the Arabidopsis rwt43 transformant indicate the opposite. Upon transition from
low to high irradiance, no lag in photosynthesis occurred in these plants, even though RuBP levels were
much lower than in wild-type plants because of the
abnormally high activation state of Rubisco at low irradiance (Zhang et al., 2002). Accordingly, NPQ did not
increase in the rwt43 transformant after transition from
low to high irradiance, presumably because the rapid
increase in CO2 assimilation provided an outlet for the
excess photochemical energy.
Studies with mutants and transgenic plants have
shown that Rca is essential for keeping Rubisco active
(Salvucci et al., 1985; Mate et al., 1993; Jiang et al.,
1994). However, our results comparing growth and
photosynthesis in the Arabidopsis wild type and the
rwt43 transformant indicate that the only obvious
penalty for not regulating Rubisco in response to irradiance was possibly a slight reduction in the rate of
CO2 assimilation at ambient CO2 (Zhang et al., 2002).
However, since Rubisco content was not reduced in
the rwt43 transformant, it is unclear why the rate of
CO2 assimilation was reduced in these plants. Perhaps
the imbalance between the rates of RuBP consumption
and regeneration in the rwt43 transformant, particularly at
low irradiance (Supplemental Table S2), exert a feedback
effect on the overall turnover rate of the Calvin cycle,
making the cycle RuBP limited (Mott et al., 1984).
Biomass accumulation was enhanced under an alternating light regime in the Arabidopsis rwt43 transformant but not in wild-type plants. However, no
enhancement occurred when dark was used instead of
low irradiance. These results were consistent with the
different responses of Rubisco activation to low irradiance versus dark (Supplemental Table S1). Taken
together, these data suggest that the faster rate of photosynthetic induction in the transformant translated into
a greater carbon gain under fluctuating light conditions.
However, developmental factors associated with the
alternating light regime or longer photoperiod cannot
be totally excluded from consideration. For example,
Rca expression is driven by a constitutive promoter in
the Arabidopsis rwt43 transformant, whereas Rca expression in the wild type exhibits a strong circadian
rhythm (Liu et al., 1996). This difference in the nature of
the Rca promoter, as well as differences in the 39 untranslated region between the native Rca gene and the
rwt43 transgene, are likely to affect the expression of
Rca under the fluctuating light regime (DeRidder et al.,
2012) and might influence the expression of other photosynthetic genes. Still, the data presented here suggest a
new strategy for enhancing photosynthetic performance
under variable light environments based on altering
the regulatory properties of Rca to increase the rate of
photosynthetic induction.
MATERIALS AND METHODS
Plant Material and Growth Conditions
Arabidopsis (Arabidopsis thaliana) wild type (ecotype Columbia) and the
transgenic lines rwt43 and Tob-DAt(S2) have been described previously (Zhang
et al., 2002; Kumar et al., 2009). Arabidopsis rwt43 is a transformant of the rca
mutant that expresses only the Arabidopsis 43-kD b-isoform of Rca (Zhang
et al., 2002). Arabidopsis Tob-DAt(S2), formerly called Arab-Tob, is also a
transformant of the rca mutant, but this line expresses a chimeric b-Rca that is
similar in sequence to the tobacco (Nicotiana tabacum) Rca except for inclusion
of the Rubisco recognition domain from Arabidopsis (Kumar et al., 2009).
Arabidopsis plants were grown in controlled-environment chambers in air
at 23°C with a 10-h photoperiod and an irradiance of 175 mmol m22 s21. Plants
were initially grown in a CO2-enriched atmosphere (2,900 mL L21 CO2) at 23°C
with an 11-h photoperiod and an irradiance of 80 mmol m22 s21. After 2 weeks,
plants were transferred to the conditions described above. Camelina (Camelina
sativa ‘Robinson’) plants were grown in air at 23°C/18°C with a 12-h photoperiod and an irradiance of 400 mmol m22 s21. Tobacco ‘Petit Havana’ SR-1 plants
were grown in air at 28°C/23°C with a 16-h photoperiod and an irradiance of
300 mmol m22 s21.
All plants were grown from seed in soil mixture and watered as needed with
nutrient solution (Carmo-Silva and Salvucci, 2011) at either full strength (for
tobacco) or one-half strength (for all other species). Measurements were conducted on fully expanded leaves of 4- to 5-week-old plants of Arabidopsis and
camelina and 5- to 6-week-old plants of tobacco.
Rca Activity in Leaf Extracts
The assay for measuring Rca activity in leaf extracts is described in detail
elsewhere (Carmo-Silva and Salvucci, 2011). Briefly, leaf discs were excised
from intact plants 3 to 4 h after the beginning of the photoperiod, immediately
frozen in liquid nitrogen, and stored at 280°C. Freshly frozen leaf material
was rapidly extracted at 4°C, desalted by centrifugal gel filtration, and then
assayed to determine the rate of ATP-dependent reactivation of the inactive
Rubisco-RuBP complex at 25°C. Activation reactions contained 90 mM Tricine-
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Carmo-Silva and Salvucci
NaOH, pH 8.0, 10 mM NaHCO3, and 30 mM MgCl2 plus or minus 5 mM ATP
and an ATP-regenerating system consisting of 5 mM phosphocreatine and 100
units mL21 phosphocreatine kinase. Where stated, ATP was replaced by a
mixture of 5 mM ADP plus ATP at a ratio of 0.11 or 0.33 in the absence of ATPregenerating system. Measurements of ADP concentration at the end of the
reaction showed that the ratio of ADP to ATP and the total adenine nucleotide
pool did not change markedly during the assay. The rate of Rca activity was
calculated as described previously (Carmo-Silva and Salvucci, 2011).
Rubisco Activation State in Leaf Extracts
Leaf discs (0.5 cm2) were excised from intact plants in the light, floated on
25 mM MES-NaOH, pH 5.5, at 25°C, and flushed with a humidified gas stream
(380 mL L21 CO2 and 21% oxygen). For the irradiance treatments, the discs
were first illuminated at high irradiance (1,000 or 1,800 mmol m22 s 21) for
20 min followed by low irradiance (30 mmol m22 s21) for 1 h. For the highirradiance treatment, discs were exposed to high irradiance (1,000 or 1,800
mmol m22 s21) for an additional 20 min after the low-irradiance treatment.
Discs were snap frozen in liquid nitrogen, and samples consisting of two discs
each were stored at 280°C. Frozen leaf discs were rapidly extracted and
assayed as described previously (Carmo-Silva and Salvucci, 2012). The activation state was calculated as the initial activity divided by the average total
activity at high irradiance, multiplied by 100.
Purification and Assay of Rca
Recombinant Arabidopsis, tobacco, and engineered tobacco proteins were
purified after expression in Escherichia coli as described previously (van de Loo
and Salvucci, 1996). Point mutations were introduced into the tobacco complementary DNA (cDNA) sequence by directed mutagenesis (van de Loo and
Salvucci, 1996). For construction of the Tob-DAt(n=17) mutant protein, the tobacco oligonucleotide sequence was engineered to change 17 amino acids in
the sequence to the corresponding residues in Arabidopsis Rca. A cDNA with
these changes was synthesized commercially (Blue Heron Biotechnology) and
expressed in E. coli. The rate of ATP hydrolysis by the purified enzymes was
determined by the amount of inorganic phosphate released as described
previously (Barta et al., 2011). Inorganic phosphate was determined by the
method of Chifflet et al. (1988). The total adenine nucleotide concentration in
the assay was 5 mM, presented at a ratio 0.00, 0.11, or 0.33 ADP/ATP. Reaction
times were adjusted to ensure that the amount of ATP consumed in the reaction was no more than 10%.
Gas-Exchange and Chlorophyll
Fluorescence Measurements
Gas exchange of attached leaves was measured at air levels of CO2 (380 mL
L21) and oxygen, at the temperatures and irradiance indicated in the figure
legends, using a portable infrared gas analyzer (LI-6400; LI-COR). For measurement of the induction of CO2 assimilation, plants were initially placed at
high irradiance (1,000 or 1,800 mmol m22 s21) to fully activate Rubisco. After
20 min, the irradiance level was decreased to 30 (Arabidopsis) or 50 (camelina
and tobacco) mmol photons m22 s21 (i.e. low irradiance) and maintained at this
level for the times indicated in the figure legends. Induction was initiated by
instantaneously increasing the irradiance level back to the high level and
measuring the rate of CO2 assimilation over time. Rates of CO2 assimilation
were corrected for lags in stomatal conductance using the response of the rate
of CO2 assimilation to the intercellular CO2 concentration (Woodrow and
Mott, 1989). Relaxation times were calculated as described by Woodrow and
Mott (1989). A PAM-2000 chlorophyll fluorometer (Heinz-Walz) was used to
measure the induction of NPQ (Carmo-Silva and Salvucci, 2012).
Miscellaneous
Protein concentration in leaf extracts was determined by the method of
Bradford (1976). The Rubisco used for determining RuBP levels was purified
from Arabidopsis leaves as described previously (Carmo-Silva et al., 2011a).
Statistical Analysis
All values are expressed as means 6 SE of all samples analyzed for
each treatment (n as indicated). Statistical comparisons between different
treatments were made using either Student’s t tests (for two treatments) or
ANOVA followed by the Holm-Sidak method for multiple pairwise comparisons (for more than two treatments). P , 0.05 was considered to represent
statistical significance.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Multiple sequence alignment of the C-terminal
extension of the Rca a-isoform from several vascular plant species and a
moss.
Supplemental Figure S2. Response of CO2 assimilation rate to irradiance
in wild-type Arabidopsis and the rwt43 transformant.
Supplemental Figure S3. Aboveground biomass accumulation in wildtype Arabidopsis and the rwt43 transformant grown under different
light regimes.
Supplemental Table S1. Effect of irradiance on the activation state of
Rubisco in wild-type Arabidopsis and the rwt43 transformant after initial deactivation in the dark.
Supplemental Table S2. Effect of irradiance on the levels of triose phosphate, Fru-1,6-bisP, and RuBP in wild-type Arabidopsis and the rwt43
transformant.
ACKNOWLEDGMENTS
We thank Nancy Parks (U.S. Department of Agriculture, Agricultural
Research Service) for expert technical assistance with cDNA cloning and protein expression and Matthew Hilton (Arizona State University) for his assistance with the metabolite measurements.
Received December 23, 2012; accepted February 14, 2013; published February
15, 2013.
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