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10.1146/annurev.arplant.55.031903.141714

Annu. Rev. Plant Biol. 2004. 55:85107

doi: 10.1146/annurev.arplant.55.031903.141714
c 2004 by Annual Reviews. All rights reserved
Copyright

METABOLIC CHANNELING IN PLANTS

Brenda S.J. Winkel
Annu. Rev. Plant Biol. 2004.55:85-107. Downloaded from arjournals.annualreviews.org
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Department of Biology and Fralin Biotechnology Center, Virginia Tech, Blacksburg,

Virginia 24061-0346; email: winkel@vt.edu

Key Words enzyme complex, metabolon, structural biology, phenylpropanoid

pathway
Abstract The organization of cooperating enzymes into macromolecular complexes is a central feature of cellular metabolism. A major advantage of such spatial
organization is the transfer of biosynthetic intermediates between catalytic sites without
diffusion into the bulk phase of the cell. This so-called metabolic channeling offers
unique opportunities for enhancing and regulating cellular biochemistry. Studies in a
number of plant primary and secondary metabolic systems continue to contribute to our
understanding of the nature and importance of this phenomenon. This article reviews
advances in four systems: the cysteine synthase complex, the Calvin cycle, cyanogenic
glucoside biosynthesis, and the phenylpropanoid pathway. Each of these systems is
providing new evidence for the importance of enzyme organization in cellular biochemistry as well as exclusive insights into the molecular basis of enzyme complex
assembly. This review also explores current prospects for understanding metabolon
structure, assembly, and biological function.
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
RECENT PROGRESS IN METABOLON RESEARCH
IN PLANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Cysteine Synthase Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Calvin Cycle and the Role of Small Proteins
in Enzyme Complex Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dhurrin Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The Phenylpropanoid Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Other Examples of Enzyme Complexes in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . .
FUTURE PERSPECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Structural Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Information from High-Throughput or Global Interaction-Detection
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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INTRODUCTION
The concept of metabolic channeling and the compartmentalization of cellular
biochemistry has a long and distinguished history (reviewed in 90). This view of
highly organized metabolism dates back to the 1930s and 1940s and is based on
now-classical experiments in biochemistry, cell biology, and genetics. Examples
include the isolation of aggregates of tricarboxylic acid (TCA) cycle enzymes by
David Green in the 1940s, (29) and the finding that the reaction intermediate of
the bifunctional tryptophan synthase system does not diffuse into solution, described by Charles Yanofsky in 1958 (108). In 1960 Marko Zalokar published
a remarkable study showing that, when ultracentrifugation was used to stratify
the intracellular contents of Neurospora hyphae, all measurable enzyme activities were associated with organelles, membranes, or the cytoskeleton, with little
or no protein present in the aqueous or soluble fraction (Figure 1) (110). Extensive experimental and theoretical work over the past 45 years has extended these
early findings to a widely accepted view of cellular metabolism involving the assembly of cooperating enzymes into complexes or metabolons, often associated
with structural elements within the cell (reviewed in 74, 90). One of the central
implications of this concept is that it affords metabolic processes the ability to
channel intermediates between two or more active sites without mixing into the
bulk phase of the cell (reviewed in 61, 89). Metabolic channeling can thus easily be
envisioned as a means to attain high local substrate concentrations, regulate competition between branch pathways for common metabolites, coordinate the activities
of pathways with shared enzymes or intermediates, and sequester reactive or toxic
intermediates.
Although this concept has been controversial at times (12, 76, 106), the importance of metabolons in cellular biochemistry is strongly supported by metabolic
control theory (11, 21, 51) as well as experimental evidence from the analysis
of biochemical pathways in various organisms. Among the best-characterized
metabolons are the tryptophan synthase, pyruvate dehydrogenase, and glycine
decarboxylase systems, the TCA and Calvin cycles, the enzymes of glycolysis and
fatty acid oxidation, the proteasome, and the machinery of macromolecular (i.e.,
fatty acid, nucleic acid, and protein) biosynthesis. All of these systems involve

Figure 1 Schematic of Neurospora hypha centrifuged at 35,000 rpm in a SW39 rotor. FAT,
fat; VAC, vacuoles; CYT, enchylema; NUC, nuclei; MIT, mitochondria; ERG, ergastoplasm;
GLY, glycogen. Reprinted with permission from Zalokar (110).

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high-affinity, stable enzyme associations that are most amenable to experimental

analysis; however, there is mounting evidence that even pathways that were once
believed to consist largely of soluble proteins appear to be subject to this type of
subcellular structuring (74). Such short-lived or dynamic complexes, which involve weak or transient interactions among enzyme components, may dissociate
and reform in response to the metabolic status of the cell, thereby providing a rapid
and powerful mechanism for regulating cellular biochemistry.
A number of important examples of both stable and dynamic enzyme complexes have emerged from the study of plant metabolism. This article reviews
advances in four of these systems, two in primary metabolism (the enzymes of
cysteine biosynthesis and the Calvin cycle) and two in secondary metabolism
(the cyanogenic glucoside and phenylpropanoid pathways). Each of these systems
is providing new evidence for the importance of enzyme organization in cellular biochemistry as well as unique insights into the molecular basis of enzyme
complex assembly. Central themes include (a) the role of protein interactions,
involving either consecutive or nonconsecutive enzymes, in regulating metabolic
activity, (b) the use of metabolons to overcome a surprising lack of substrate specificity in many enzymes, (c) the concept of information transfer between proteins,
(d) indications that small proteins may be involved in guiding the assembly of
enzyme complexes, and (e) evidence that there is still a need to define even the
basic biochemical steps in some of these pathways. This review will also explore
current prospects for understanding metabolon structure, assembly, and biological
function. Future directions are being fueled by rapid advances in structural biology
paired with existing and emerging technologies to identify protein partners and
characterize macromolecular assemblies in vitro and in situ.

RECENT PROGRESS IN METABOLON RESEARCH

IN PLANTS
The Cysteine Synthase Complex
The interaction of enzymes that catalyze sequential biochemical reactions has
long been seen as a way to bring cooperating active sites into close proximity,
a hallmark of metabolic channeling. In many cases, experimental evidence has
borne this out, for example, for the TCA cycle enzymes, citrate synthase and
malate dehydrogenase (69), and the enzymes of dNTP synthesis (reviewed in
61). However, studies of the cysteine synthase complex in plants and bacteria are
challenging the idea that all interactions between sequential enzymes are for the
purpose of metabolic channeling.
The synthesis of cysteine from serine is catalyzed by two enzymes, serine
acetyltransferase (SAT), which catalyzes the entry step from serine metabolism,
and O-acetylserine(thiol)-lyase (OAS-TL), the second and terminal enzyme in
the pathway (Figure 2). These enzymes have been found in eubacteria, fungi,
plants, and some archaebacteria, but not in mammals, which can only synthesize

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Figure 2 Schematic of cysteine biosynthesis. SAT and OAS-TL refer to the two
enzymes in this pathway, serine acetyltransferase and O-acetylserine(thiol)-lyase.

cysteine indirectly from methionine (36, 52, 60, 72). The association of these enzymes was first reported in the bacterium Salmonella typhimurium (53). Studies in
several plant species indicate that the two enzymes exist as multimeric complexes
located in mitochondria, chloroplasts, and the cytoplasm. Evidence for the interaction of SAT and OAS-TL has come from copurification of the enzymes from rape
(70), chives (71), and spinach (16), reconstitution of the complex with recombinant
forms of the cytoplasmic enzymes from watermelon (82) and, most recently, the
use of a yeast two-hybrid system and surface plasmon resonance (SPR) to analyze
interactions between the mitochondrial enzymes from Arabidopsis (8, 9).
It has long been assumed that the association of SAT and OAS-TL serves
to channel O-acetylserine between the two active sites, preventing diffusion and
enhancing catalytic efficiency. However, work by Droux et al. (17) in Arabidopsis
has uncovered a different purpose for this interaction: that OAS-TL binds SAT,
not to serve as part of a metabolic channel, but as a structural and/or regulatory
subunit that has significantly reduced catalytic activity. The bulk of the OAS-TL
activity appears to be associated with the uncomplexed form of the enzyme. These
conclusions are based on kinetic analysis of the free and bound enzymes, showing a
strong positive effect of complex formation on SAT activity and negative effect on
the activity of OAS-TL, the finding that the O-actylserine intermediate is released
into the bulk solution and thus apparently not channeled between the two active
sites, and the observation that there is a large excess of OAS-TL in mitochondria,
chloroplasts, and the cytoplasm. The negative impact of complex formation on
OAS-TL activity has also been demonstrated in E. coli (63). This interpretation
of the interaction of the two enzymes is also consistent with the observation that
the two substrates of OAS-TL destablize the complex in both plant and bacterial
systems (8, 17, 53). Maximal in vitro activity of spinach SAT occurs only when
OAS-TL is present in vast excess, again consistent with a dual role for this protein,
including one as a catalytically inactive enhancer of SAT activity (81). Evidence
that this is also the case in vivo comes from the recent finding that overexpression

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of a wheat OAS-TL gene enhances the ability of transgenic tobacco plants to

tolerate oxidative stress, apparently by elevating the overall levels of OAS-TL and
increasing the capacity to generate cysteine and glutathione (109).
The cysteine synthase bienzyme system thus provides a new perspective on
enzyme associations. Although suggestive of a role in metabolic channeling, the
interaction of enzymes that catalyze sequential biosynthetic reactions may have a
different purpose. The dual role of OAS-TL in cysteine biosynthesis underscores
the idea that enzymes can play both structural and catalytic roles, as is also the
case for proteins that associate to form a metabolic channel between active sites.
However, it alerts us to the possibility that enzymes may not necessarily carry out
both functions at the same time and that the structural role can be a regulatory
one. It is interesting to speculate that the regulatory mechanism of the cysteine
synthase system evolved from an ancestral bienzyme complex formed for the
purpose of metabolic channeling, and that additional examples of consecutive
pathway enzymes using analogous regulatory mechanisms remain to be identified.

The Calvin Cycle and the Role of Small Proteins

in Enzyme Complex Formation
New insights into both the function and mechanism of assembly of enzyme complexes are coming from studies of the Calvin cycle in both plants and the green
alga Chlamydomonas. This metabolic system supports a role for interactions between enzymes that catalyze consecutive and those that catalyze nonconsecutive
reactions in the regulation of metabolic activity. It also provides some of the first
evidence for the involvement of small, noncatalytic proteins in the assembly of
a multienzyme system, a mechanism that appears to have been highly conserved
over the evolution of this ancient pathway.
Evidence that the enzymes of the Calvin cycle are organized as a multienzyme
complex comes from a number of studies, performed largely with spinach and pea
chloroplasts, that also suggest that this complex is loosely associated with thylakoid membranes near the sites of ATP and NADPH synthesis (Figure 3) (24,
24, 78, 95). There is further evidence that carbonic anhydrase is located at the
periphery of this complex, providing CO2 directly to ribulose 1,5-bisphosphate
carboxylase-oxygenase (RuBISCO) (39). Assembly of the complex not only enhances the activities of component enzymes like RuBISCO, but also the regulation
of phosphorebulokinase (PRK) by reduced thioredoxin, a major mechanism for
light activation of the pathway (25). Recent work with Chlamydomonas indicates
that PRK and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are regulated
by NADP(H) when they are part of the complex, but by NAD(H) when they are not
(28). Spatial organization of this enzyme system thus appears to affect metabolic
function, not only by providing a mechanism for enhanced efficiency by channeling intermediates as shown in the past, but by modifying the activity and regulation
of the individual enzymes.
Surprisingly, the Calvin cycle also appears to be regulated by interaction of
PRK and GAPDH, even though these enzymes catalyze nonconsecutive reactions

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Figure 3 Schematic of the Calvin cycle. Superscripts refer to evidence for the presence of
some or all of the enzymes in a complex copurification (1) (24), (2) (78), and (6) (39); protein interaction (3) (4) and (4) (101); and colocalization at membranes (5) (3, 51). Phosphoribulokinase,
ribulose-1,5-bisphosphate carboxylase/oxygenase, and sedoheptulose-1,7-bisphosphate are
unique to the Calvin cycle; the others are shared with glycolysis or the pentose phosphate
pathway.

in this pathway. The activation of both enzymes depends not only on reduction of
the active sites by thioredoxin, but also on dissociation of a complex composed of
PRK or GAPDH homo-oligomers, or of hetero-oligomers of the two enzymes together with a small chloroplast protein known as CP12 (85). The NADPH-mediated
dissociation of PRK/CP12/GAPDH appears to represent a highly conserved mechanism for light activation of Calvin cycle activity. CP12 is a small protein with
homology to the C terminus of the B subunit of GAPDH that has been found in
plants, algae, and the cyanobacterium Synechocystis. Its function appears to be
highly conserved because the Synechocystis protein can replace pea CP12 in the
pea PRK/CP12/GAPDH complex (103).
The PRK/CP12/GAPDH complex has also been extensively characterized in
Chlamydomonas, where the situation appears to be slightly different than in
cyanobacteria and higher plants. This may be due, in part, to the fact that green
algae lack the GAPDH B subunit, to which CP12 has homology. Initial work by
Lebreton, Gontero, and colleagues showed that Chlamydomonas PRK is strongly

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Figure 4 Model for assembly of the PRK/CP12/

GAPDH complex of C. reinhardtii. Reprinted with permission from Graciet et al. (27).

activated upon binding to GAPDH and that the two substrates of PRK, ATP and
ribulose 5-phosphate, destabilize or stabilize the complex, respectively (6, 55). It
was proposed that GAPDH imparts an imprinting effect that causes PRK to transiently adopt a highly active conformation after dissociation (55); PRK appears to
have a similar effect on GAPDH (28). Recently, this group has used SPR to measure binding constants for these proteins (27). These results support a model for the
assembly of the PRK/CP12/GAPDH complex in which CP12 first associates with
GAPDH, changing the conformation of the enzyme and allowing association with
PRK, and eventually resulting in formation of a native complex composed of two
dimers of PRK, two tetramers of GAPDH, and two monomers of CP12 (Figure 4).
It is clear that the Calvin cycle is characterized by complex enzyme interactions,
the significance of which remains to be fully characterized. It also remains to be
determined how much similarity exists between cyanobacteria, green algae, and
higher plants in the overall regulation of this system via the PRK/CP12/GAPDH
complex. It should be noted that many of the enzymes of the Calvin cycle also
function in glycolysis, a system in which extensive evidence for protein interactions
and substrate channeling has also been described (and debated) in many different
organisms (recent examples include 7, 10) so that the observations in each of these
pathways may have a much broader relevance.

Dhurrin Biosynthesis
In contrast to the high-affinity protein interactions typical of enzyme systems like
the cysteine synthase complex and the Calvin cycle, secondary metabolism often
appears to be characterized by transient, low-affinity enzyme interactions. These

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types of dynamic complexes are inherently more difficult to study, but evidence
supporting their fundamental importance is emerging from the work of Birger
Mllers group on the biosynthesis of dhurrin in sorghum. Dhurrin is a member
of an important class of secondary compounds, the cyanogenic glycosides, which
are produced by a wide variety of plant species, including sorghum, cassava,
cherry, and almond, for protection against herbivores (reviewed in 23). These
compounds also function in nitrogen storage and in the control of germination. In
defense, toxic cyanide is produced upon degradation of the glycoside by enzymes
that are stored in a different subcellular compartment and brought together upon
tissue disruption, as occurs during herbivory. Remarkably, cyanogenesis can be
engineered into otherwise acyanogenic plants by coexpressing just three enzymes
(Figure 5); this was recently demonstrated by using the three sorghum genes needed
for the synthesis of dhurrin to engineer resistance to the flea beetle, Phyllotreta
nemorum, in transgenic Arabidopsis (97).
The lability and high toxicity of biosynthetic intermediates provide a strong
rationale for the existence of metabolic channeling via close association of active
sites in the dhurrin pathway. The earliest indications that dhurrin synthesis involves
channeling date back 25 years to the experiments of Mller & Conn (67), who
used radiolabeled compounds to demonstrate channeling of N-hydroxytyrosine
and p-hydroxyphenylacetonitrile in sorghum microsomes. It is now known that
these are reaction intermediates for two bifunctional cytochrome P450 enzymes,
CYP79A1 and CYP71E1 (Figure 5), (47, 88) and that the pathway is similarly organized in other cyanogenic plants (73). However, the intermediate, phydroxyphenylacetaldoxime, derived from tyrosine by the activity of CYP79A1,
exchanges freely with exogenously added aldoxime, suggesting the absence of
channeling between these two enzyme systems. Moreover, microsomes were unable to carry out the final glycosylation step and it was not possible to cross-link
the glucosyltransferase with the microsomal components, indicating at best a loose
association of the terminal enzyme with the other two.
The use of fusion proteins carrying variants of green fluorescent protein, together with the ability to examine this system in intact plant cells using confocal
laser scanning microscopy, is providing new evidence for the existence of a dhurrin
metabolon involving all three enzymes. Tattersall et al. (98) found that fusion of
yellow and cyan fluorescent protein to the two P450 enzymes dramatically reduces
dhurrin production, despite having no effect on catalytic activity. This suggests that
the fusion partners cause steric hindrance that interferes with enzyme interaction
and substrate channeling. These workers also showed that the soluble glucosyltransferase is localized to distinct domains of the endoplasmic reticulum (ER)
membrane, but only when both P450 enzymes are present, providing the first evidence for the association of the terminal enzyme with the rest of the system.
Such a loose association of the glucosyl transferase is also similar to what has
been observed for these enzymes in other systems (46). The scenario of a dhurrin
metabolon consisting not only of multifunctional components but of interacting enzymes is consistent with the observations of very low levels of intermediates in this

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Figure 5 Schematic of the biosynthesis of the cyanogenic glucoside, dhurrin,

in Sorghum bicolor. The pathway is catalyzed by two multifunctional microsomal cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble UDPGglucosyltransferase (sbHMNGT). Cyanogenesis has been engineered in Arabidopsis
by coexpressing these three enzymes (97). Redrawn from Kahn et al. (48).

pathway, both in the original experiments using sorghum microsomes (67) and
more recently in engineered Arabidopsis (97). It is further supported by the observation that, despite the generally broad substrate specificities of glucosyltransferases and the presence of 112 predicted family 1 UDP-glucose glucosyltranferases in Arabidopsis, sorghum UGT85B1 is still required for dhurrin biosynthesis
in transgenic Arabidopsis plants. The dhurrin pathway thus provides compelling
evidence for the importance of even low-affinity enzyme interactions in the production of plant secondary metabolites and underscores the need to study these types
of enzyme systems in the context of the cell. The system should also provide an
excellent experimental model for determining the biochemical and physiological

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significance, if any, of dynamic versus stable enzyme interactions in cellular

metabolism.

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The Phenylpropanoid Pathway

In 1974, Helen Stafford (91, 92) proposed that the enzymes of phenylpropanoid
metabolism are organized as one or more enzyme complexes. In this system
metabolic channeling was envisioned to provide a mechanism not only for sequestering unstable or toxic intermediates, but for controlling flux among the multiple branch pathways that often function concurrently in the same cell. Phenylpropanoid metabolism, which is largely unique to plants, transforms phenylalanine
into a variety of important secondary products, including lignins, sinapate esters,
stilbenes, and flavonoids, the latter also incorporating carbon from acetate (reviewed in 13, 33, 34, 105). These compounds are of fundamental importance to
the plant cell, functioning as phytoalexins, UV sunscreens, pigments, signaling
molecules, and major structural components. Although complex, it is among the
best-studied metabolic systems in plants.
There is strong experimental support for spatial organization of phenylpropanoid
enzymes in the cytoplasm of plant cells. Studies in a wide range of species have generated evidence for the co-ordinate expression of genes and enzymes, channeling
of labeled intermediates, membrane association of operationally soluble enzymes,
and physical interaction of component enzymes. There is also new support for the
existence of a metabolic channel between phenylalanine ammonia-lyase (PAL)
and cinnamate 4-hydroxylase (C4H) in the general phenylpropanoid pathway (77)
and between enzymes of the isoflavonoid branch pathway in legumes (35). There is
also evidence for colocalization of the first two enzymes of the flavonoid pathway
in Arabidopsis root cells (83). These data are consistent with the long-standing
model in which many, and perhaps all, enzymes of phenylpropanoid metabolism
constitute one or more ER-associated multienzyme complexes. Still, the exact nature of this organization and its biochemical and physiological significance remain
to be established.
Several possibilities have been suggested regarding the precise nature of this organization. For example, Arabidopsis contains three isoforms of 4-coumarate:CoA
ligase (4CL) that have different substrate preferences and expression patterns, and
that appear to represent two evolutionarily divergent classes (19). It is possible that
these enzymes function in distinct metabolons, each dedicated to producing a particular class of phenylpropanoids (Figure 6). The slight preference of AtCL1 and
AtCL2 for caffeic acid over p-coumarate and of AtCL3 for the latter was initially
suggested to be consistent with this; however, caffeic acid now appears to represent
a minor route to lignin, with p-coumarate serving as a common intermediate in
both lignin and flavonoid biosynthesis (reviewed in 38). Perhaps this difference
reflects an evolutionary history that includes alternative pathways to lignin. More
compelling is gene expression data, which suggests a role for AtCL1 in lignin synthesis, with a more limited role for AtCL2, perhaps also in the synthesis of other

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Figure 6 Schematic of branch pathways of phenylpropanoid metabolism, organized as

individual metabolons. Enzymes that function in multiple branches are in the same shade
of gray, enzymes unique to specific pathways are in white, and those known to be present
as multiple isoforms are indicated with an asterisk (PAL, 3 genes; 4CL, 2 genes; FLS, 4
genes plus 2 pseudogenes). The P450 hydroxylases, C4H, C3H, F5H, and F30 H, are shown
as integral membrane proteins; these have been postulated to provide scaffolds for the selfassembly of enzyme complexes in this and other systems.

phenolics. In contrast, the gene encoding AtCL3 exhibits an expression pattern

consistent with a role in flavonoid production. Thus it is quite possible that each of
these isoforms represents a specific (nonshared) component of an enzyme complex
leading to lignins or flavonoids in Arabidopsis, as suggested by Ehlting et al. (19).
Distinct isoforms of PAL, the first enzyme in the pathway, have also been associated with proanthocyanidin and lignin biosynthesis in Populus tremuloides
(quaking aspen) (49). PAL is also encoded by a small gene family in Arabidopsis
(102), whereas C4H, a cytochrome P450 enzyme that catalyzes the second step
in the pathway, appears to be encoded by a single gene (66). However, expression analysis suggests that it will be more difficult to assign different PAL isoforms to branch pathways of phenylpropanoid metabolism. Rick Dixons group
has used transgenic tobacco to generate new evidence for channeling between

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specific forms of PAL and C4H (77). This group has also shown that the two forms
of PAL in tobacco are located either at the ER, in the case of PAL1, or in the
cytoplasm, for PAL2 (1). However, PAL2 can be induced to localize to the ER by
overexpression of C4H, and this localization can be reversed by overexpression of
PAL1. This points to protein interactions between the membrane P450 enzyme,
C4H, and the two PAL enzymes, with a particularly strong association for PAL1.
These findings point to distinct roles for the two PAL isoforms, reflected in differential interactions with C4H. Another point of interest is that one of the two
isoforms of NADPH:cytochrome P450 reductase in Arabidopsis has been linked
with phenylpropanoid metabolism (65). It is possible that this protein is involved in
localization and perhaps also in assembly of phenylpropanoid enzyme complexes
in conjunction with P450 enzymes such as C4H.
Evidence for channeling in the isoflavonoid branch pathway was recently reported by the Dixon group. Among the most compelling is the observed differences
in the in vitro and in vivo specificities of isoflavone O-methyltransferase from Medicago sativa (alfalfa) and localization of this operationally soluble enzyme to the
ER upon elicitation of the isoflavonoid pathway (35, 56). These findings, together
with evidence for channeling of radiolabeled intermediates, have led to the suggestion that the methyltransferase is closely associated with the integral membrane
cytochrome P450 enzyme, isoflavone synthase (IFS), facilitating rapid methylation
and stabilization of its product. These researchers have also shown that efforts to
engineer isoflavonoid synthesis in Arabidopsis by introducing IFS into a flavonoid
overexpressing line or in conjunction with alfalfa chalcone isomerase (CHI) results
in a disproportionate decrease in flavonoid production, which cannot be explained
by a shift in flux alone (57). One possible explanation offered for this outcome was
that the membrane-localized IFS might interfere with the assembly of metabolic
complexes dedicated to flavonoid biosynthesis, which are also anchored to the ER
by a cytochrome P450, flavonoid 30 -hydroxylase (F30 H).
Several examples of the association of flavonoid enzymes or endproducts with
as-yet-unidentified structures in a variety of plant tissues have been reported recently. These include the association of a GFP-labeled C4H from poplar with
mobile, elongated (100 nm 600 nm) fluorescent organelles, suggested to be
subdomains of the ER, in hypocotyls and cotyledon epidermal cells of transgenic Arabidopsis seedlings (79). Large (300-nm diameter) uniformly fluorescent bodies were also observed in the guard cells of these seedlings, which were
speculated to be protein storage vacuoles and the fluorescence possibly an artifact of C4H::GFP overexpression. Localization of UDP-glucose:flavonol 20 - and
50 -O-glucosyltransferases in electron-dense particles has also been reported in
Chrysosplenium americanum (American gold saxifrage) leaves (54), and PAL
and chalcone synthase (CHS) have been found in as-yet-unidentified spherical
compartments in Primula kiwensis (Kew primrose) (87). In addition, our laboratory described the colocalization of CHS and CHI in electron-dense particles
(200 nm diameter) in Arabidopsis root cells, which were not observed in a tt7
mutant lacking the P450 hydroxylase, F30 H, the proposed membrane anchor for

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the flavonoid pathway (83). There are similarities in the observed localization with
that of vacuolar targeting proteins and the Bp-80 putative vacuolar sorting receptor. Although a connection is not yet clear, these observations are reminiscent of
the fluorescent bodies reported in maize BMS cells ectopically expressing the P
transcription factor (30). These structures are somewhat larger, on the order of 10
50 M, but have been speculated to contain the endproducts of phenylpropanoid
branch pathways. Taken together, these observations suggest that further characterization of these intriguing observations could provide new insights into how the
synthesis and deposition of phenylpropanoid products are organized within plant
cells.
As in most other systems, the biochemical and physiological relevance of
metabolic channeling in phenylpropanoid metabolism remains to be established.
There are hints that enzyme localization may be important, for example, in auxin
transport or the response to wounding (D. Saslowsky & B. Winkel, unpublished
data; 83). The role of different isozymic forms in controlling flux in response to
different developmental or environmental cues also remains to be determined. The
accumulated wealth of information on phenylpropanoid metabolism is one of its
great strengths as a model for studying enzyme organization. However, it remains
a complex system in which the configurations of branch pathways continue to be
refined and even rewritten, as with the recent surprising identification of shikimate
and quinate intermediates in the route to lignins (reviewed in 38) and a newly
defined role for anthocyanidin reductase in proanthocyanidin biosynthesis (107).

Other Examples of Enzyme Complexes in Plants

There are countless other metabolic systems in plants for which evidence of enzyme
complex assembly exists. One of the most well-established is the glycine decarboxylase system, which provides a unique example of how the physical proximity
of active sites in an enzyme complex can lead to efficient catalysis of sequential
reactions (Figure 7) (reviewed in 15). This system is linked to serine biosynthesis
via a mechanism that has been proposed to involve channeling of the folate cofactor. New examples also continue to emerge, for example, from the work of Panicot
et al. (75), who recently demonstrated interactions among enzymes of polyamine
biosynthesis in Arabidopsis, suggesting the existence of a metabolon comprising
at least the final two steps in this pathway. Plant metabolism clearly continues to
provide a rich source of material for elucidating enzyme organization and its role
in facilitating and regulating cellular biochemistry.

FUTURE PERSPECTIVES
Structural Biology
After decades of accumulating evidence for the existence and importance of enzyme complexes in cellular metabolism, the ability to model these systems in three

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dimensions is only now approaching reality. In particular, significant progress has

been made for the phenylpropanoid pathway, where the past four years have seen
a tremendous effort by Joe Noels group and collaborators, who have solved highresolution structures for CHS (22), CHI (41), caffeic acid/5-hydroxyferulic acid
3/5-O-methyltransferase (112), and chalcone and isoflavone O-methyltransferases
from alfalfa (111). The structure for anthocyanidin synthase (ANS) from Arabidopsis has now also been solved by Christopher Schofield and coworkers (104).
These structures, as well as homology models based on these (Figure 8) and other
more distantly related proteins, as has been done for 4CL (86), are providing
important insights into the mechanism and evolution of enzyme function, and
promise to help define the molecular basis for enzyme interactions and metabolic
channeling.
CHS is of broad interest because it is a member of a superfamily of plant
condensing enzymes known as the type III polyketide synthases, homologs of
which have now also been found in several bacterial genomes (5, 68). These
enzymes are more distantly related to the type I and II polyketide synthases and
to the -ketoacyl synthases and thiolase enzymes of fatty acid metabolism. The
architecture of the CHS homodimer has shown how each monomer contributes to
formation of the two active sites, how substrate preference is likely determined
by residues in the coumaroyl binding pocket, and how the cyclization pocket
may limit the number of acetate additions and control the stereochemistry of the
endproduct; the predicted mechanism has some similarities, but also significant
differences, with the more distantly related enzymes (22). Structural analysis has
also identified a CoA-binding tunnel in CHS that allows substrates to access the
buried active site. This arrangement provides further support for the importance of
channeling in this system because it points to the exchangeability of CHS reaction
intermediates, for example, allowing for the action of chalcone reductase on the
polyketide intermediate during isoflavonoid biosynthesis (22). In addition, sitedirected mutagenesis of CHS, together with structural determinations for several
related enzymes, has provided key experimental support for the predictions made
from structural data (40, 44, 45, 94) and is now used to engineer these enzymes
for the biosynthesis of novel products (42).
The other flavonoid enzyme structures are each noteworthy in their own right.
CHI is of special interest because it represents a structure unique to higher plants,
and site-directed mutagenesis of the active site suggests that the related enzymes
from other plants use a common reaction mechanism (41, 43). On the other hand,
ANS is a member of the ubiquitous oxoglutarate-dependent dioxygenase family,
for which only one other structure has been solved to date, for isopenicillin synthase from Aspergillus nidulans (80). The flavonoid pathway contains three other
dioxygenases, flavanone 3-hydroxylase, flavone synthase, and flavonol synthase.
Analysis of the determinants of substrate specificity in ANS is particularly intriguing in light of evidence that this enzyme can catalyze the same reaction as flavonol
synthase in vitro (99). Finally, structural analysis of three methyltransferases that
function in lignin and isoflavonoid biosynthesis has provided the first glimpses

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into the architecture of an important class of enzymes with broad functions in

plant metabolism (111, 112). This work helps explain the much broader substrate
specificity of the lignin pathway enzyme relative to the other two and provides a
new approach to engineering lignin biosynthesis, for example, to reduce the environmental impact and cost of wood processing, or to enhance the digestibility of
forage crops.
This new structural information may well be the Rosetta stone for deciphering
the biochemical regulation of phenylpropanoid metabolism by making it possible
to develop three-dimensional models of enzyme organization. It also provides a
molecular understanding of the remarkable lack of substrate selectivity of many
enzymes that points to enzyme channeling as an important means of control. It is
increasingly clear that enzyme structures can be used to deduce reliable structures
for homologs from other species, providing access to a wealth of biochemical
information and the unique insights that can be gained from protein evolution.
There is some reason to believe that protein interaction domains in enzymes, as in
transcription factors, may be highly conserved. In the phenylpropanoid pathway,
for example, it has been possible to complement Arabidopsis CHS, CHI, and dihydroflavonol reductase mutants with the corresponding maize genes (14), and the
operationally soluble PAL enzyme from bean localizes to microsomal membranes
in tobacco, similar to tobacco PAL2 (77). Structures of related enzymes proposed
to function in distinct enzyme complexes, such as the methyltransferases leading
to G and S monolignols in the lignin pathway, could be used to identify domains
required for interaction with distinct pathway components (18). At the same time,
information on the characteristics of protein interaction domains is accumulating
at a rapid pace, including remarkable new insights from computational approaches.
One notable example, from Ruth Nussinovs group, is the use of a multiple structure comparison algorithm, MUSTA, to analyze proteins with well-characterized
interaction domains (58). This approach has identified three residues, Trp, Phe,
and Met, that are highly conserved in binding sites but not elsewhere on the protein
surface and thus provide new hallmarks for a priori prediction of protein-protein
interfaces. By developing an understanding of proteins as a whole, the combined information from structural, experimental analysis and computational analysis may
open the doors to engineering not only the catalytic properties of enzymes, but
their ability to interact with other components of existing or introduced enzyme
complexes.
Tractable methods for analyzing protein interactions are becoming the limiting
factor in the goal to generate three-dimensional models of enzyme complexes.
Currently, applying SPR analysis to measure the kinetics of interaction between
recombinant proteins and using tandem affinity purification to copurify proteins
from plant cells provides some of the newest and most promising approaches. Intriguing examples of the use of fluorescence resonance energy transfer (FRET) to
monitor protein interactions in planta were published recently (37, 50), although
experience with dhurrin pathway enzymes, in which the fluorescent protein tags interfered with enzyme interactions (98), suggests that this method may have inherent

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limitations for the study of metabolic complexes. Methods for structural characterization of macromolecular assemblies are also urgently needed. One promising
technology is small-angle neutron scattering, which can provide structural information for large assemblies, on the scale of viral particles, in solution (reviewed
in 96). Some evidence indicates that this technology could also be a powerful tool
for deducing the architecture of multienzyme complexes (C. Dana, S. Kreuger, &
B. Winkel, unpublished data).

Information from High-Throughput or Global

Interaction-Detection Methods
A number of emerging technologies offer the promise of studying metabolism
from a perspective that does not rely on a full understanding of biochemical pathways and is free of preconceived notions of metabolic organization. Computational methods, such as critical analysis of large datasets generated by genome
sequencing or the use of microarrays, can be quite reliable in predicting enzyme
associations in known complexes, such as the glycine decarboxylase system (100).
Although this is a relatively stable complex, interactions between the four yeast
enzymes were not detected using tandem affinity purification, and yet the corresponding genes could be linked through three other lines of evidence: association
in operons of seven divergent prokaryotes, similar phylogenetic distributions (the
genes are either all present or, in 17 genomes, all absent), and coexpression under diverse conditions based on microarray data. This type of approach will be
most useful in defining complexes involving stable interactions, enzymes of ancient evolutionary origin, and ones that are expressed at high levels. However,
as these analyses are refined and the datasets increase in size and number, computational approaches should become increasingly powerful tools for confirming
and predicting a wide range of interdependent protein systems. At the same time,
new experimental methods for high-throughput screening of interacting proteins
or domains are emerging. A recently described assay based on the association of
reconstituted murine dihydrofolate reductase with a fluorescent probe suggests the
possibility of screening libraries expressed in plant protoplasts for the presence of
interacting proteins or peptides (93). Technologies such as phage, bacterial cell
wall, and yeast display could prove particularly useful for identifying interacting
domains from high-diversity libraries of peptide sequences (reviewed in 59). Together, these global approaches should provide much-needed tools for confirming,
predicting, and characterizing a wide range of protein systems, including here-tofore unknown catalytic and structural components of metabolic complexes, and
should even help identify connections between pathways.
There is also the prospect of extending the analysis of enzyme complexes,
and metabolic channeling in particular, to another level: the in vivo imaging of
metabolites. Fehr et al. (20) built on the chamelion concept (64) using FRET
with a modified version of the bacterial periplasmic maltose binding protein to
generate a fluorescent nanosensor for maltose levels in living yeast cells. Binding

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of maltose causes a conformational change in the sensor, bringing together two

different GFP variants bound to its N and C terminus and resulting in a change
in fluorescence emission. When introduced into cells, this system allows direct
monitoring of changes in cytosolic maltose concentrations resulting from changes
in the extracellular supply. One can easily envision applying such a system to
directly monitor metabolic channeling (i.e., the diffusion of intermediates into the
cellular milieu) and to examine the effects on channeling of mutations in interaction
domains or in proteins required to assemble or anchor a complex within the cell.
The key to making this a generally useful approach will be to identify or develop
proteins with the desired binding specificities.
It is clear that our understanding of metabolic organization continues to mature, even as the biological implications of this phenomenon remain elusive. The
use of cryoelectron tomography by Wolfgang Baumeisters group to visualize the
interior of the eukaryotic cell (31, 62) [remarkably reminiscent of David Goodsells cartoon renderings of the E. coli cell (26)] is generating a renewed sense
of urgency to understand how the cellular machinery operates in such a crowded
environment. Our immediate challenge is to take full advantage of the many powerful new approaches to this long-standing and fundamental problem in cellular
biochemistry.
ACKNOWLEDGMENTS
The author gratefully acknowledges the National Science Foundation (grants
DMB9304767, MCB9808117, and MCB0131010) and the U.S. Department of
Agriculture (grant 200103371) for support of her laboratorys work on flavonoid
metabolism. Chris Dana generated the revised version of the Arabidopsis CHS
homology model shown in Figure 8.
The Annual Review of Plant Biology is online at http://plant.annualreviews.org

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Figure 7 Schematic of reactions involved in oxidative decarboxylation and deamination of glycine in plant mitochondria. Reprinted with permission from Douce et al.
(15).

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Figure 8 Homology model of the Arabidopsis CHS monomer based on the structure of alfalfa CHS2 (22). Active site residues (C169, F220, N342, and H309) are
shown in yellow, the methionine (M142) that contributes to the active site in the
adjacent subunit of the holoenzyme is in red. The image was generated using Swiss
PDB Viewer version 3.7 (32) and rendered with POV-Ray version 3.1
(http://www.povray.org). Adapted from (84).

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Annual Review of Plant Biology

Volume 55, 2004

CONTENTS
Annu. Rev. Plant Biol. 2004.55:85-107. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF ESSEX on 06/13/10. For personal use only.

AN UNFORESEEN VOYAGE TO THE WORLD OF PHYTOCHROMES,

Masaki Furuya

ALTERNATIVE NAD(P)H DEHYDROGENASES OF PLANT

MITOCHONDRIA, Allan G. Rasmusson, Kathleen L. Soole,
and Thomas E. Elthon

DNA METHYLATION AND EPIGENETICS, Judith Bender

PHOSPHOENOLPYRUVATE CARBOXYLASE: A NEW ERA OF
STRUCTURAL BIOLOGY, Katsura Izui, Hiroyoshi Matsumura,
Tsuyoshi Furumoto, and Yasushi Kai

METABOLIC CHANNELING IN PLANTS, Brenda S.J. Winkel

RHAMNOGALACTURONAN II: STRUCTURE AND FUNCTION OF A
BORATE CROSS-LINKED CELL WALL PECTIC POLYSACCHARIDE,
Malcolm A. ONeill, Tadashi Ishii, Peter Albersheim, and Alan G. Darvill

23
41

69
85

109

NATURALLY OCCURRING GENETIC VARIATION IN ARABIDOPSIS

THALIANA, Maarten Koornneef, Carlos Alonso-Blanco, and
Dick Vreugdenhil

141

SINGLE-CELL C4 PHOTOSYNTHESIS VERSUS THE DUAL-CELL (KRANZ)

PARADIGM, Gerald E. Edwards, Vincent R. Franceschi,
and Elena V. Voznesenskaya

173

MOLECULAR MECHANISM OF GIBBERELLIN SIGNALING IN PLANTS,

Tai-ping Sun and Frank Gubler

PHYTOESTROGENS, Richard A. Dixon

DECODING Ca2+ SIGNALS THROUGH PLANT PROTEIN KINASES,
Jeffrey F. Harper, Ghislain Breton, and Alice Harmon

PLASTID TRANSFORMATION IN HIGHER PLANTS, Pal Maliga

SYMBIOSES OF GRASSES WITH SEEDBORNE FUNGAL ENDOPHYTES,
Christopher L. Schardl, Adrian Leuchtmann, Martin J. Spiering

197
225
263
289
315

TRANSPORT MECHANISMS FOR ORGANIC FORMS OF CARBON AND

NITROGEN BETWEEN SOURCE AND SINK, Sylvie Lalonde,
Daniel Wipf, and Wolf B. Frommer

341

vii

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CONTENTS

REACTIVE OXYGEN SPECIES: METABOLISM, OXIDATIVE STRESS,

AND SIGNAL TRANSDUCTION, Klaus Apel and Heribert Hirt
THE GENERATION OF Ca2+ SIGNALS IN PLANTS,
Alistair M. Hetherington and Colin Brownlee

BIOSYNTHESIS AND ACCUMULATION OF STEROLS, Pierre Benveniste

HOW DO CROP PLANTS TOLERATE ACID SOILS? MECHANISMS OF
ALUMINUM TOLERANCE AND PHOSPHOROUS EFFICIENCY,
Annu. Rev. Plant Biol. 2004.55:85-107. Downloaded from arjournals.annualreviews.org
by UNIVERSITY OF ESSEX on 06/13/10. For personal use only.

Leon V. Kochian, Owen A. Hoekenga, and Miguel A. Pineros

VIGS VECTORS FOR GENE SLIENCING: MANY TARGETS,

MANY TOOLS, Dominique Robertson
GENETIC REGULATION OF TIME TO FLOWER IN ARABIDOPSIS THALIANA,
Yoshibumi Komeda

373
401
429

459
495
521

VISUALIZING CHROMOSOME STRUCTURE/ORGANIZATION,

Eric Lam, Naohiro Kato, and Koichi Watanabe

537

THE UBIQUITIN 26S PROTEASOME PROTEOLYTIC PATHWAY,

Jan Smalle and Richard D. Vierstra

555

RISING ATMOSPHERIC CARBON DIOXIDE: PLANTS FACE THE FUTURE,

Stephen P. Long, Elizabeth A. Ainsworth, Alistair Rogers,
and Donald R. Ort

591

INDEXES
Subject Index
Cumulative Index of Contributing Authors, Volumes 4555
Cumulative Index of Chapter Titles, Volumes 4555

ERRATA
An online log of corrections to Annual Review of Plant Biology
chapters may be found at http://plant.annualreviews.org/

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