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Plant Physiology and Biochemistry 41 (2003) 523–532
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Review
Regulation of pyruvate, orthophosphate dikinase by ADP-/Pi-dependent
reversible phosphorylation in C3 and C4 plants >
Chris J. Chastain a,*, Raymond Chollet b
b
a
Department of Biology, Minnesota State University-Moorhead, Moorhead, MN 56563, USA
Department of Biochemistry, University of Nebraska-Lincoln, George W. Beadle Center, Lincoln, NE 68588-0664, USA
Received 21 November 2002; accepted 7 February 2003
Abstract
Pyruvate, orthophosphate dikinase (PPDK, E.C. 2.7.9.1) is a cardinal carbon-assimilating, stromal enzyme of the C4 photosynthetic
pathway. Like several other photosynthetic pathway enzymes, its activity is strictly and reversibly regulated by light. This regulation is
conferred by the PPDK regulatory protein (RP), a bifunctional protein kinase/phosphatase that catalyzes the ADP-/Pi-dependent, reversible
phosphorylation of an active-site threonine residue. In this minireview, we highlight how plastidic PPDK in leaves and developing seeds of C3
plants is regulated in an identical manner as C4 PPDK via a putative C3-RP isoform. Additionally, we also detail the progress in research
concerning C4 RP, since this highly unusual regulatory enzyme was last reviewed nearly two decades ago.
© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
Keywords: C4 photosynthesis; C3 plant; PPDK; PPDK regulatory protein; Protein phosphorylation/dephosphorylation; Pyruvate, Pi dikinase; RP
1. Introduction
Historically speaking, plant pyruvate, orthophosphate
dikinase (PPDK; E.C. 2.7.9.1) was initially discovered in C4
leaves, where it is an abundant mesophyll-chloroplast enzyme involved in C4 photosynthesis [16]. A major emphasis
was placed on gaining an understanding of its now wellelucidated regulation via site-specific reversible phosphorylation of a target Thr residue [16,34], because of its role as a
cardinal stromal enzyme in the C4-photosynthetic pathway.
In contrast, the presence of PPDK in C3 plants, where it is of
non-photosynthetic function and usually of low abundance,
Abbreviations: CAM, Crassulacean acid metabolism; CCCP, carbonyl
cyanide m-chlorophenlyhydrazone; DCMU, 3-(3,4-dichlorophenyl)-1,1dimethylurea; MV, methyl viologen; PEP, phosphoenolpyruvate; PEPC,
PEP carboxylase; Pi, inorganic phosphate; PPDK, pyruvate, orthophosphate
dikinase; PPT, plastidic PEP/Pi translocator; PS, Photosystem; RP, PPDK
regulatory protein.
>
This minireview is dedicated to Dr. Pierre Gadal on the occasion of his
retirement from the Université de Paris-Sud (Centre d’Orsay) for his numerous important discoveries related to metabolic enzymes and their regulation
in plants.
* Corresponding author.
E-mail address: [email protected] (C.J. Chastain).
© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
DOI: 10.1016/S0981-9428(03)00065-2
was not established until much later. Further, research into
the function and regulation of PPDK in C3 plants has received only scant attention and it remains for the most part
unexplored.
This minireview will highlight the recently documented
regulation of PPDK in C3 leaves via reversible protein phosphorylation [14] and its implication for putative function(s).
Additionally, we will detail the significant progress concerning the C4 PPDK regulatory protein (RP), a bifunctional
protein kinase/phosphatase that catalyzes the most unusual,
ADP-/Pi-dependent reversible phosphorylation of C4 PPDK,
since this topic was last reviewed in depth nearly two decades
ago [10,16].
2. C4 PPDK and its regulation by the bifunctional,
PPDK regulatory protein
2.1. C4 PPDK
PPDK in C4 plants catalyzes the regeneration of the primary CO2 acceptor phophoenolpyruvate (PEP) in the chloroplast stroma of leaf mesophyll-cells. It is maximally active
as a homotetramer of ~95-kDa subunits and dissociates into
largely inactive dimers and monomers when subjected to
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C.J. Chastain, R. Chollet / Plant Physiology and Biochemistry 41 (2003) 523–532
Fig. 1. Partial reactions and overall catalysis by PPDK showing the reversible, 3-step formation of PEP from pyruvate, ATP, and Pi: (a) pyrophosphorylation of
the active-site His residue (e.g., His-458 in maize C4 PPDK) by the b- and c-phosphates of ATP; (b) subsequent transfer of the c-phosphate to Pi yielding PPi
and PPDK-HisP; and (c) phosphorylation of pyruvate by PPDK-HisP to form PEP and free enzyme (E-His) [11,24]). The dots in partial reactions (a) and (b)
indicate a complex of PPDK-HisPP, AMP, and Pi.
cold temperatures in vitro [6]. As one of two potentially
rate-limiting enzymes in the C4 pathway [19], it has been
extensively researched and its biochemical properties well
characterized ([11,16] and references therein). The overall
PPDK catalytic mechanism is a complex and readily reversible, 3-step reaction sequence that deploys free (E-His), pyrophosphoryl (E-His-PP), and phosphoryl (E-His-P) forms
of an essential His residue in the active-site domain (e.g.,
His-458 in maize C4 dikinase [8,13] (Fig. 1 and [11,24]).
2.1.1. Regulation of PPDK in C4 photosynthesis: the C4
PPDK regulatory protein
As a potentially rate-limiting enzyme in the C4 pathway,
the synchronization of PPDK activity with light availability
in vivo is essential for efficient functioning of the C4 cycle
and its coordination with the C3 pathway. This form of
regulation was established earlier by studies that measured
PPDK activity in desalted crude leaf extracts from illuminated and darkened C4 plants, with illuminated leaves possessing high PPDK activity and dark-adapted leaves showing
negligible activity ([16] and references therein). Some important clues concerning the mechanism of light-/darkregulation of C4 PPDK were provided by these simple, initial
experiments and included the observations that (a) light activation of dark-inactivated PPDK was specific for photosynthetically active radiation (i.e., red and blue peaks in a related
action spectrum) and inhibited by DCMU, and (b) incubation
of inactive PPDK in crude leaf extracts at room temperature
could in some way “reactivate” the enzyme [16]. In both
cases, the activation of inactive PPDK was ultimately attributed to dephosphorylation of an active-site threonine residue
(e.g., Thr-456 in maize C4 PPDK) [4,10,16,34]. Ensuing
from this discovery was the revelation that a bifunctional
protein kinase/phosphatase with unprecedented enzymatic
properties catalyzed this reversible phosphorylation event
[9,10] (Fig. 2). Now “nicknamed” the PPDK regulatory protein or “RP”, it is one of less than a handful of known
regulatory protein kinases or phosphatases with similarly
novel attributes. Among these is its bifunctionality, catalyzing both PPDK inactivation (phosphorylation) and activation (dephosphorylation). This is very rare as most regulatory
phosphorylation cycles have separate kinase and phosphatase enzymes [21,22,39]. Furthermore, when functioning
as a protein kinase, it uses ADP instead of ATP as the specific
phosphoryl donor. Conversely, it employs a Pi-dependent,
PPi-forming phosphorolytic dephosphorylation mechanism,
as opposed to simple hydrolysis as used by most protein
phosphatases [39]. All of these unique catalytic attributes
would perhaps imply remarkable structural properties as
well, but such knowledge has remained elusive since the
gene or cDNA for RP has not been cloned, nor has the
enzyme been reproducibly purified to homogeneity
[9,32,38], the latter precluding its cloning by conventional
Fig. 2. Simplified working model of the regulatory phosphorylation of C4
PPDK by its bifunctional RP in the mesophyll-chloroplast stroma. As a
consequence of dark-induced increases in stromal [ADP], the RP-catalyzed
kinase reaction is favored, while PPDK dephosphorylation is strongly inhibited by ADP (see Table 1). In the light, stromal ADP levels decrease due to
photophosphorylation, thus favoring the RP-catalyzed dephosphorylation
reaction and subsequent reactivation of PPDK. The strictly conserved,
catalytically essential His-P residue (Fig. 1) at the P+2 position (e.g.,
His-458 in maize C4 PPDK [13]) is also indicated. (Modified from Fig. 1,
Chastain et al. [14].)
C.J. Chastain, R. Chollet / Plant Physiology and Biochemistry 41 (2003) 523–532
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Table 1
Some important kinetic parameters of maize RP as measured in vitro [5,9,10,32]
Kinase properties
Km (ADP)
Km (PPDK-Thr)
Specific activity with PPDK-Thr
Ki (Pyruvate) a
50 µM
1.2 µM
1.5 U mg–1 RP
80 µM
Phosphatase properties
Km (Pi)
Km (PPDK-ThrP)
Specific activity with PPDK-ThrP
Ki (ADP)
Ki (PPi)
650 µM
0.7 µM
0.59 U mg–1 RP
84 µM
160 µM
a
Likely inhibits the threonyl-phosphorylation of PPDK by direct competition with RP for the requisite E-HisP reaction intermediate during catalysis by
PPDK (see Figs. 1 and 2 and [5,9]).
means. Most of what is known about the detailed enzymatic
properties of C4 RP has originated from essentially five
published studies of the maize enzyme, four of which date
back to the mid-1980s [2,5,9,32]. Some of these findings are
summarized in Table 1. Additional information on RP from
these earlier studies included (a) estimates of a monomeric
molecular mass of 45,000-48,000, as determined by sizeexclusion chromatography and one-dimensional SDS-PAGE
[9,38]; (b) pH-dependent changes in aggregation state
(dimeric at pH 7.5, tetrameric at pH 8.3) [9]; and (c) in vitro
evidence for physically distinct active-site domains for its
unique protein kinase and protein phosphatase functions
[10,32].
2.1.2. C4 PPDK active-site Thr and His replacement
studies: insights into RP function and the PPDK
inactivation mechanism
More recently, further insight into the functional properties of C4 RP has been gained by selective substitutions of the
maize C4 PPDK active-site His residue (His-458) and the
proximal target Thr residue (Thr-456) for RP [12,13]. These
results are summarized inTable 2. Among the more informative Thr-456 substitutions were Ser and Tyr. These showed
that Ser was functionally interchangeable with the wild-type
Thr residue, and could readily serve as a target for ADPdependent phosphorylation by RP, while Tyr could not. This
documents that RP is functionally a Ser/Thr kinase and
implies that it is potentially related to the Ser/Thr family of
protein kinases, rather than the “dual-specificity” family that
targets all three hydroxyamino acids. Another informative
substitution was the replacement of the catalytic His with
Asn, a chemically related but non-phosphorylatable residue.
As expected, this substitution produced a catalytically incompetent PPDK, but it also rendered the enzyme resistant to
phosphorylation by exogenous RP, despite harboring the
adjacent target Thr. The striking inability of this H458N
mutant enzyme to undergo phosphorylation provided direct
support for earlier biochemical studies which suggested that
RP’s protein kinase function has an absolute substrate requirement for the His-P form of the target enzyme (see Figs.
1 and 2 and [7,8]). Subsequent substitutions of the regulatory
Thr residue with Asp, Glu, and Phe provided insight into the
mechanism of how PPDK is inactivated when the enzyme is
specifically phosphorylated at position 456. From previous
work, it was demonstrated that the phosphorylation of a
single active-site Thr residue completely abolished enzyme
activity [7,34]. One plausible means by which catalysis can
be entirely eclipsed by a single phosphorylation event is
suggested by the close proximity of this regulatory Thr residue to the catalytic His. In all the C4 (and C3) plant PPDK
genes sequenced to date, the position of the regulatory Thr is
conserved as the second residue N-terminal of the catalytic
His (Fig. 3). Introduction of a negatively charged, dianionic
(2-) phosphoryl group proximal to the catalytic His would be
expected to interfere via electrostatic repulsion with the
negatively charged substrates (pyruvate, ATP, Pi) bound to
the flanking remote domains of PPDK, which pivot to the
central catalytic domain harboring the essential His [24].
Such a phosphorylation-based regulatory mechanism is not
Table 2
Summary of the target Thr residue (Thr-456) and central catalytic His
(His-458) replacement studies using maize recombinant C4 PPDK [12,13]
Substitution
%WT PPDK activity
Thr456Val
Thr456Ser
Thr456Tyr
Thr456Phe
Thr456Glu
Thr456Asp
His458Asn
98
111
6
1
0
0
0
Phosphorylation by exogenous
maize-leaf RP
No
Yes
No
No
No
No
No
Fig. 3. Aligned active-site sequences of representative green plant, protozoan and bacterial PPDKs showing the strict conservation of the catalytically essential His and plant regulatory Thr residues in all groups.
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C.J. Chastain, R. Chollet / Plant Physiology and Biochemistry 41 (2003) 523–532
without precedent [31], and it appears to be the most likely
scenario since replacement of the regulatory Thr with the
monoanionic (1-) side chain of Asp or Glu abolishes both
enzyme activity and phosphorylatability (Table 2). Alternatively, the inactivation mechanism may also include a steric
component in addition to an electrostatic one since substitution of Thr by Tyr or Phe (but not Val), two neutral amino
acids with bulky side chains, also dramatically inhibits dikinase activity (Table 2). However, high-resolution crystal
structures of these wild-type and mutant C4 enzymes are
needed to understand these striking Thr-456 substitution
effects with some certainty.
2.2. Regulation of the C4 PPDK regulatory protein
The competing reactions of RP must somehow be flexibly
governed for correctly adjusting PPDK activation state to the
rate of carbon flux in the C4 (and C3) cycle because it is
bifunctional, with the ability to catalyze both phosphorylation and dephosphorylation of PPDK. Some evidence to date
suggests that the mechanism providing this flexible and sensitive regulation may be a relatively simple one, involving
only light-/dark-induced changes in stromal concentrations
of the competing RP substrates, ADP and Pi, and the potent
dephosphorylation inhibitor ADP (Fig. 2,Table 1) [9]. According to this proposed mechanism, the higher ADP/ATP
ratios occurring in the dark (i.e., increased stromal [ADP])
favor the inactivation/phosphorylation reaction. Key to this
proposed mechanism is the observation that ADP is a potent
competitive inhibitor of the dephosphorylation reaction in
vitro (Table 1). This would maintain PPDK in its inactive
Thr-P state during the dark, despite moderately high levels of
stromal Pi. In the light, as stromal [ADP] decreases during its
conversion to ATP via photophosphorylation, the dephosphorylation (PPi-forming) reaction proceeds and the pool of
inactive enzyme becomes fully reactivated. Evidence supporting this working model comes from earlier studies that
examined the effects of DCMU, a PSII electron-transport
inhibitor, and CCCP, an uncoupler of photophosphorylation,
on maize C4-mesophyll-protoplast and -chloroplast PPDK
activity [28,29]. These findings showed that illumination of
mesophyll-cell preparations in the presence of DCMU or
CCCP markedly inhibited light activation of PPDK, and this
was correlated with lowered stromal ATP concentrations in
the light. Yet to be reconciled with this simple “regulation by
adenylates” model are a pair of related studies that examined
light/dark changes in in vivo PPDK activation state with
respect to in vivo changes of mesophyll-cell and -chloroplast
[ADP] [33,41]. In both studies, the observed light-induced,
10-fold change in maize leaf PPDK activity was not highly
correlated with these measured changes in [ADP]. Conversely, direct regulation of RP does not appear to involve
any post-translational modifications (e.g., stromal redoxregulation). This view is supported by a study that examined
RP activity after it was rapidly extracted from dark-adapted
or illuminated maize leaves [38]. RP activity from these
leaves showed no preferential direction in catalysis, i.e.,
having equivalent relative competence in the in vitro phosphorylation or dephosphorylation of PPDK, regardless of the
light/dark pretreatment of the parent leaves. Furthermore, the
ratio of rapidly extracted, competing RP activities was also
shown to be independent of pH utilized for extraction and
assay. These observations indicate that a post-translational
regulatory mechanism, e.g., covalent modification, changes
in stromal pH, is not evident under conditions in which RP
displays distinct in vivo regulation of its competing reactions. Likewise, stromal redox state, a well-known regulatory
mechanism for many stromal enzymes (via the
ferredoxin/thioredoxin system), also has been shown to have
no influence on RP regulation in organello or in vitro
[16,29,38]. Thus, the mechanism by which the bifunctional
activities of C4 RP are regulated in vivo can likely be attributed simply to light-/dark-induced changes in stromal levels
of ADP, and to a lesser extent Pi, but see [33,41] for a
contrasting view.
3. Regulation of C3 PPDK by reversible
phosphorylation
3.1. PPDK in C3 plants
As is the case with other C4 pathway enzymes, such as
PEP carboxylase (PEPC) and NADPH-malate dehydrogenase, PPDK is also present in C3 plants, and, likewise, this
isoform is not believed to participate directly in photosynthesis. The dikinase found in C3 plants is highly homologous to
the C4 enzyme, with respect to its primary structure and
biochemical properties ([17,25,27,35] see Fig. 3). In most C3
plant tissues and organs, PPDK is a ubiquitous but lowabundance enzyme localized in both the cytoplasm [1,27,30]
and chloroplast [1,14]. The single example where PPDK is in
abundance comparable to C4 leaves is in developing cereal
seeds, where it is expressed in a developmentally regulated
manner [20,30]. The gene for PPDK in C3 plants, as in C4
species, may be present in two copies, as it is in rice, with
each gene-copy representing a similar but distinct isogene
[27]. However, in the C3 dicot Flaveria pringlei, it is apparently a single-copy gene as revealed by Southern analysis
[35]. Analysis of the Arabidopsis genome also indicates that
PPDK is a single-copy gene in this model C3 plant. In those
C3 and C4 species with two PPDK genes, one copy encodes
an exclusive, cytoplasmically localized isoform, while the
other gene can be expressed either as a plastid-targeted dikinase or a cytoplasm-targeted PPDK, via a unique “dual”
promoter configuration [25,36]. The latter of these two isogenes is commonly referred to as the “C4-type” PPDK gene,
since the stromal PPDK isoform utilized in the C4 pathway is
expressed from this type of structural gene. C3 plants with a
single PPDK gene-copy possess the “C4-dual promoter” type
gene, but lack the second cytoplasm-only targeted isogene.
Although PPDK in C3 plants occurs with ubiquity (e.g., in
roots, stems, leaves, seeds), its precise metabolic functions
are unknown but are likely to be multifaceted due to its
C.J. Chastain, R. Chollet / Plant Physiology and Biochemistry 41 (2003) 523–532
frequent presence in both the cytoplasm and plastid. One of
the major difficulties in elucidating a specific metabolic
role(s) for PPDK in C3 plants is that its relatively low abundance in most organs precludes the following turnover of
substrates or products by conventional radiotracer techniques. Furthermore, catalysis by PPDK is readily reversible,
with the potential for catalyzing the formation of either PEP
in one direction or pyruvate (and ATP) in the other (Fig. 1).
For example, in contrast to the direction of catalysis by
dikinase in C4 photosynthesis, endobacteria and amitochondriate protozoa utilize PPDK in the pyruvate/ATP-forming
direction [3]. Lastly, PEP carboxykinase, also present in
plant cells, reversibly catalyzes PEP synthesis as well, further complicating analysis of PPDK function. Our insights
into a plausible role for the plastidic isoform of C3 PPDK are
discussed in Section 4, but a more detailed understanding of
both cytoplasmic and plastidic PPDK functions in plants will
require transgenic approaches utilizing gene knock-outs
and/or genetically engineered, selective up-down-regulation
of endogenous PPDK activity.
3.2. Regulation of C3-leaf PPDK by reversible
phosphorylation
Early attempts at detecting regulation of PPDK in C3
leaves utilized an approach based on measuring PPDK activity in crude leaf extracts prepared from light- and darkadapted tissues, or in one report, lysate from isolated intact
chloroplasts [1]. Studies using this indirect approach for
assessing in vivo PPDK regulation were largely inconclusive
due to the problems inherent in assaying a low-abundance
enzyme, such as C3 PPDK, via routine NADH oxidationPEPC-coupled spectrophotometric methods. Furthermore,
earlier studies assaying PPDK activity in crude C3-leaf extracts were not able to discriminate between cytoplasmic
dikinase activity (non-light-regulated, i.e. constitutively active) and chloroplastic activity (reversibly light-activated), as
both isoforms intermingle in whole tissue extracts. However,
more recent studies utilizing C3 transgenic plants with abundantly expressed chloroplastic PPDK were able to overcome
these difficulties by producing more accurately measurable
levels of PPDK activity [18,26,37]. These studies were far
more suggestive of a light-/dark-modulated mechanism regulating PPDK in C3 chloroplasts. For example, in transgenic
tobacco lines that overexpressed the M. crystallinum CAMPPDK isogene, a 4-5-fold increase in PPDK leaf protein was
accompanied by only a 1.5-fold increase in extractable
PPDK activity [37]. A similar study of transgenic Arabidopsis overexpressing maize chloroplastic C4-PPDK also
showed a large increase in leaf PPDK protein but with less of
an increase in extractable PPDK activity [26]. A plausible
explanation for the relatively low recovery of extractable
PPDK activity from these transgenic PPDK overexpressers is
that much of the increased stromal dikinase protein could
have been inactivated in vivo via regulatory phosphorylation.
A recent study of transgenic rice overexpressing the maize
C4-PPDK transgene (chloroplast-targeted) produced more
527
convincing evidence by specifically examining PPDK activity from light- and dark-adapted transgenic leaves [18].
When the variable of light vs. dark conditions was controlled,
as in this rice study, a distinct and readily measurable,
several-fold increase in PPDK activity extracted from lightvs. dark-adapted transgenic leaf tissue was observed. These
highly discernible, light-/dark-induced differences in extractable dikinase activity led the authors to conclude that an
endogenous RP-like activity was mediating this up-/downregulation of the heterologous C4 PPDK in their transgenic
rice lines. We have recently provided direct, complementary
evidence for the presence of an endogenous C3-RP isoform
and the associated light-/dark-regulation of chloroplastic
PPDK in C3 plants by reversible phosphorylation [14]. In
brief, we have determined that the regulation of C3-leaf
PPDK by reversible protein phosphorylation is highly analogous to the well understood C4 PPDK regulatory system. Key
to providing this evidence has been the development and
utilization of a synthetic phosphopeptide-generated antibody
that is specific for PPDK phosphorylated at its strictly conserved (Fig. 3), target Thr residue [13,14]. Using this affinitypurified, polyclonal antibody, we have developed a highly
exacting and sensitive immunological method that readily
detects dark-/light-modulated phosphorylation of PPDK in
C3 leaves and chloroplasts in a broad spectrum of species
(Fig. 4 and [14]). We also established that PPDK phosphorylation in C3 leaves is readily reversible and kinetically similar
in vivo to that occurring in C4 plants (Fig. 5 and [14,16,33]).
3.3. Evidence for a C3-isoform of RP in C3 chloroplasts
In C4 plants, RP is localized, together with its target
enzyme, in chloroplasts of leaf mesophyll-cells. This also
appears to be the case in C3 plants as evidence to date would
suggest. One line of supporting data comes from experiments
utilizing isolated, intact spinach (C3) chloroplasts in which
the mechanism catalyzing dephosphorylation of chloroplas-
Fig. 4. PPDK in C3 leaves undergoes light-/dark-induced reversible phosphorylation. Displayed are immunoblots of C3-leaf soluble proteins probed
with phosphospecific, PPDK-ThrP PPDK antibody. Arrows indicate the
band corresponding to the ≈95-kDa PPDK monomer as estimated by molecular mass standards. Leaves were dark-adapted for 3 h (+ dark lanes) and
then reilluminated for 1 h at ~800 µmol photons m–2 s–1 (+ light lanes) prior
to extraction. Each lane contained 100 µg soluble protein. (Data are adapted
from Fig. 2, Chastain et al. [14].)
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C.J. Chastain, R. Chollet / Plant Physiology and Biochemistry 41 (2003) 523–532
Fig. 5. Comparative kinetic analysis of light-/dark-regulated changes in
PPDK phosphorylation state in maize (C4) vs. F. pringlei (C3) leaves. Each
time point represents the relative image intensity of the PPDK-ThrP bands
on immunoblots as a percent of that in a dark-adapted leaf (2 or 3 h). Light
exposure was initiated at ~800 µmol photons m–2 s–1. (Data are adapted from
Figs. 3 and 4, Chastain et al. [14].)
tic PPDK-ThrP was found to be inherently similar to the C4
RP mechanism [14]. This conclusion was ascertained by
immunoblot analysis of PPDK-ThrP dephosphorylation
upon selective inhibition of PSII- and PSI-dependent electron transport, while illuminating spinach chloroplasts in the
presence of DCMU (PSII inhibitor) or MV (PSI alternative
electron acceptor). The data from these in organello experiments, summarized in Fig. 6, indicate that PSII function is
required for dephosphorylation of PPDK-ThrP, while PSI
Fig. 6. Inhibition of Photosystem II, but not Photosystem I terminal electron
transport, impairs light-induced dephosphorylation of PPDK-ThrP in isolated, intact spinach chloroplasts. (A-C) Duplicate immunoblots of stromal
extracts prepared from intact chloroplasts incubated in the presence or
absence of 20 µM DCMU (A), 100 µM MV (B), or 2 mM potassium Pi (C)
for 10 min in the light or dark. Immunoblots were probed with either
PPDK-ThrP antibody (above) or general PPDK antibody (below). The lanes
labeled as “control” represent the PPDK phosphorylation state prior to
experimental manipulation of the intact chloroplasts isolated from the darkadapted (1.5 h) parent leaves. (Data are adapted from Fig. 6, Chastain et al.
[14].)
electron transport beyond the iron-sulfur centers FX, FA and
FB is not. This reveals insight into the C3 PPDK phosphoregulatory mechanism in two important ways. First, impairing PSII-dependent electron transport indirectly results in the
inhibition of non-cyclic photophosphorylation and, thus, increased levels of stromal ADP. Second, inhibition of terminal
electron transfer from PSI results in impairment of the subsequent reduction of stromal thioredoxin and NADP, the
latter being directly required for C3 cycle activity. That lightinduced dephosphorylation of spinach chloroplast PPDK-
C.J. Chastain, R. Chollet / Plant Physiology and Biochemistry 41 (2003) 523–532
ThrP was inhibited by blocking PSII activity (+DCMU), but
not terminal electron transport from PSI (+MV) (Fig. 6A,B)
strongly supports the view that a C3-RP isoform is responsible for phosphoregulation of C3-chloroplast PPDK. Thus,
these in organello observations are entirely consistent with
the working model of C4 RP regulation, i.e., the direction of
RP catalysis is governed largely by stromal levels of ADP
and Pi (Section 2.2 and Fig. 2) and not a chloroplast redoxrelated mechanism. Further supporting this view is the observation that when Pi, a requisite dephosphorylation substrate
for RP, is experimentally depleted during illumination of C3
chloroplasts, PPDK-ThrP dephosphorylation is inhibited
(Fig. 6C). A final line of supporting evidence that a C3-RP
isoform is chloroplast-localized, as it is in C4 plants, is
demonstrated by comparative in vitro RP assays of spinach
leaf extracts vs. spinach chloroplast lysates. This comparative analysis, shown in Fig. 7A-C, indicates that the ADPdependent, PPDK-phosphorylating activity derived from a
desalted spinach-leaf extract (containing both cytosolic and
chloroplastic components) is of chloroplast origin. This important conclusion is based on correlating the relative enrichment level of the Rubisco (large subunit) protein, a reliable
stromal marker enzyme, with that of the protein-kinase activity of RP in a leaf extract vs. a chloroplast lysate (Fig. 7B,C).
Finally, we utilized a similar in vitro RP assay to demonstrate
that spinach chloroplast lysate contains a Pi-dependent,
PPDK-ThrP dephosphorylating activity (Fig. 7D). Thus,
both requisite RP activities can be demonstrated in vitro in
this model C3 chloroplast.
3.4. Regulatory phosphorylation of C3 PPDK
in developing cereal seeds
Although PPDK is found in all organs of a C3 plant, its
potential regulation via reversible protein phosphorylation
has been examined exclusively in leaves and chloroplasts. In
this minireview, we present unpublished findings from our
laboratory which demonstrate that phosphoregulation of C3
PPDK (i.e., phosphorylation at the active-site regulatory Thr)
also occurs in developing rice seeds (Fig. 8). As discussed
above, PPDK has been shown previously to be an abundant
enzyme in developing cereal seeds [20]. The data in Fig. 8
support these earlier findings in that both PPDK protein and
activity (data not shown) in immature rice seeds were found
to be at levels rivaling those in C4 leaves (i.e., approx. 30% of
a typical C4 leaf). At this early developmental stage, when
PPDK protein and activity are present at peak levels (10-15 d
post-pollination), only traces of PPDK-ThrP could be detected (Fig. 8A,B). However, as seed maturation progressed
(20-30 d post-pollination), total PPDK protein and activity
(data not shown) declined precipitously, with a corresponding increase in phosphorylated/down-regulated PPDK. At
seed maturity (40 d post-pollination), the amounts of total
PPDK protein and phospho-PPDK protein were shown to
converge. From these preliminary data, we hypothesize that
the majority of the PPDK measured at peak levels (10-15 d
post-pollination) is the cytoplasmically localized isoform,
529
Fig. 7. Comparative analysis of spinach RP activity extracted from whole
leaves and isolated intact chloroplasts. (A) The immunoblot-based, in vitro
RP-phosphorylation assays were initiated by combining aliquots of desalted, crude spinach-leaf extract or chloroplast lysate with purified maize
recombinant (nonphospho) C4-PPDK in the presence or absence of 1 mM
ADP. Aliquots of the in vitro phosphorylation reactions, containing 1.75 µg
C4 PPDK, were electrophoresed in SDS-PAGE gels, and the resulting
immunoblots probed with PPDK-ThrP antibody. In the two -ADP lanes, the
faint band of phospho-PPDK is the result of carryover of endogenous
PPDK-ThrP present in the aliquot of the leaf extract or chloroplast lysate
used for the in vitro RP assay. (B) Relative Rubisco large subunit content in
the spinach-leaf extract vs. chloroplast lysate used in (A) as determined by
UV scanning densitometry of the SYPRO-orange stained SDS-PAGE gels
(n = 3). (C) Relative amount of RP protein-kinase activity determined in the
spinach-leaf extract vs. chloroplast lysate used in panel (A) as determined by
scanning densitometry of the PPDK-ThrP immunoblots from in vitro phosphorylation assays as shown in (A) (n = 3). (D) Spinach-chloroplast lysate
contains a Pi-dependent, PPDK-ThrP dephosphorylation activity. The immunoblots shown are of aliquots from in vitro RP-dephosphorylation assays
± Pi. The assays were identical to those in (A) except that purified maize-leaf
phospho-PPDK was employed as substrate in the presence or absence of 2
mM Pi.
while the phospho-PPDK (most prominent in mature seeds)
is the plastid-localized isoform. The much larger cytoplasmic
pool of dikinase presumably undergoes complete degradation as the seed reaches maturity, with only the stromal
PPDK-ThrP pool remaining in the mature seed. We also
detected an RP-like activity in desalted crude extracts prepared from immature rice seeds (Fig. 8C). This finding of an
ADP-dependent phosphorylation activity in immature seeds,
presumably of plastidic origin, may therefore account for the
site-specific threonyl-phosphorylation of PPDK observed
during seed development. Hence, regulatory phosphorylation of PPDK via an RP-like activity may be more ubiquitous
throughout a C3 plant, occurring in both leaves [14,18] and
other organs, such as developing seeds.
530
C.J. Chastain, R. Chollet / Plant Physiology and Biochemistry 41 (2003) 523–532
Fig. 8. Regulatory phosphorylation of PPDK in developing rice seeds. (A)
Immunoblot analysis of total soluble proteins extracted from developing rice
seeds at 5 d intervals post-pollination. Blots were probed with either antiPPDK (above) or anti-PPDK-ThrP (below) antibody. (B) Plot of total PPDK
(") and PPDK-ThrP (m) contents in developing rice seeds based on scanning densitometry of the corresponding immunoblots in (A). This plot
depicts a direct comparison of the PPDK to PPDK-ThrP proteins in ng µg–1
soluble seed protein based on a PPDK-Thr(P) standard included in one lane
of (A) (std.) n = 3. (C) Immature, developing rice seeds contain an extractable ADP-dependent, RP-like phosphorylation activity (assays performed as
described in the legend to Fig. 7A).
4. A proposed function of plastidic PPDK in C3 plants
As discussed above, elucidating specific functions of
“non-C4” PPDK is complicated by its low abundance in most
plant organs and its presence in both the cytoplasm and
chloroplast. However, the recent discovery that chloroplastic
C3 PPDK is reversibly light-regulated [14] provides new
insight into a potential function for PPDK localized in this
subcellular compartment. We propose that chloroplastic
PPDK in C3 plants functions to supplement the stromal pool
of PEP normally generated by import from the cytoplasm via
the plastidic PEP/Pi translocator or “PPT” [40]. PEP is of
pivotal importance as a substrate for the stromal shikimic
acid pathway, which in turn “fuels” aromatic amino acid
biosynthesis [15]. Furthermore, amino acid biosynthesis in
chloroplasts is a highly light-regulated process because the
various steps in the pathways are directly dependent on
photosynthetically derived energy. Thus, as part of the
broader mechanism for dark inactivation of amino acid biosynthesis, C3 PPDK is correspondingly down-regulated by
RP to perhaps prevent destabilization of the stromal pool of
PEP in the dark. Support for this interpretation comes from a
recent study of an Arabidopsis mutant lacking a functional
PPT translocator (cue1) [40]. This mutant phenotypically
shows reduced growth and vigor, and has a distinct reticulate
pattern of leaf chlorosis with pale-green cells outlying the
major veins and normal dark-green cells surrounding the
major veins [40]. All of the deleterious aspects of the cue1
mutation are reversed by overexpression of a chloroplasttargeted maize C4 PPDK transgene (genotype cue-1/PPDK)
[42]. This demonstrates that increased levels of endogenous
stromal PPDK have the potential to augment PEP imported
from the cytoplasm via the PPT. A related observation that
supports the notion that PPDK may have an augmenting role
in C3-chloroplast PEP supply is the chlorosis pattern of cue1
described above. The phenotypically normal, dark-green
cells surrounding the major veins in cue1 are notable because
of a recent report that documents an 18-fold higher PPDK
activity in cells surrounding the vascular bundles of stems
and leaf petioles of tobacco (C3) [23]. This could account for
the appearance of normal green tissue near the major veins in
the mutant cue1 leaves, with the higher amounts of PPDK
activity in this tissue largely restoring stromal [PEP] to wildtype levels. The restoration to a wild-type phenotype by
overexpression of a chloroplast-targeted C4 PPDK transgene
in cue1 supports the above hypothesis, i.e., association of
normal green cells in cue1 along the major veins with a
higher endogenous level of C3 PPDK, and suggests that
PPDK in C3 chloroplasts may indeed function to augment the
stromal pool of PEP crucially required for aromatic amino
acid biosynthesis and related phenolic metabolism.
5. Conclusions
As the research frontiers in plant carbon metabolism have
advanced beyond elucidation of the major pathways, newer
challenges have emerged in the form of identifying the many
ancillary reactions and augmenting pathways that are exclusive to plants as eukaryotes. A prime example of this is
PPDK. Well understood in its role as an abundant C4 pathway
enzyme, its function(s) as a generally low-abundance enzyme in C3 plants has remained obscure for the reasons
described in Section 3.1. The recent findings concerning C3
PPDK and its regulation summarized in this minireview
provide a renewed basis for understanding its potential functions. First of these is the finding that plastidic C3 PPDK is
regulated by reversible phosphorylation via the same mechanism (i.e., RP) as is C4 PPDK. Of similar importance is that
regulation of C3 PPDK by RP does not likely extend to the
cytoplasmic-localized isoform, which is thus presumed to be
constitutively active. Emerging knowledge of how PPDK
regulation is compartmentalized in C3 plant tissues and organs provides critical insight as this problem is eventually
explored using transgenic approaches.
Acknowledgements
This work was supported in part by Grant Nos. IBN/RUI0094497 (to C.J.C.) and MCB-9727236/MCB-0130057 (to
C.J. Chastain, R. Chollet / Plant Physiology and Biochemistry 41 (2003) 523–532
R.C.) from the U.S. National Science Foundation. R.C.’s
efforts are published as a contribution of the University of
Nebraska Agricultural Research Division (Lincoln, NE
68583, USA), Journal Series No. 13,965.
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