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Molecular Plant
Review Article
Multifaceted Regulations of Gateway Enzyme
Phenylalanine Ammonia-Lyase in the Biosynthesis
of Phenylpropanoids
Xuebin Zhang and Chang-Jun Liu*
Biological, Environmental & Climate Sciences Department, Brookhaven National Laboratory, Upton, NY 11973, USA
*Correspondence: Chang-Jun Liu (cliu@bnl.gov)
http://dx.doi.org/10.1016/j.molp.2014.11.001
ABSTRACT
Phenylpropanoid biosynthesis in plants engenders a vast variety of aromatic metabolites critically important for their growth, development, and environmental adaptation. Some of these aromatic compounds
have high economic value. Phenylalanine ammonia-lyase (PAL) is the first committed enzyme in the
pathway; it diverts the central flux of carbon from the primary metabolism to the synthesis of myriad phenolics. Over the decades, many studies have shown that exquisite regulatory mechanisms at multiple levels
control the transcription and the enzymatic activity of PALs. In this review, a current overview of our understanding of the complicated regulatory mechanisms governing the activity of PAL is presented; recent
progress in unraveling its post-translational modifications, its metabolite feedback regulation, and its
enzyme organization is highlighted.
Key words: phenylpropanoids, phenylalanine ammonia-lyase, metabolic regulation
Zhang X. and Liu C.-J. (2015). Multifaceted Regulations of Gateway Enzyme Phenylalanine Ammonia-Lyase in the
Biosynthesis of Phenylpropanoids. Mol. Plant. 8, 17–27.
INTRODUCTION
Phenylpropanoid biosynthesis converts L-phenylalanine (L-Phe)
into diverse aromatic compounds, collectively termed (poly)
phenolics. More than 8000 aromatic metabolites have been
identified in plants and categorized into different subclasses,
including the benenoids, coumarins, flavonoids/anthocyanins,
stilbenes, hydroxycinnamates, lignans, and the macromolecule
lignin (Vogt, 2010; Fraser and Chapple, 2011). This large family
of aromatic compounds fulfills numerous physiological
functions essential for plant growth, development, and plant–
environment interactions. In particular, lignin, a structural
component deposited in the vasculature, imparts mechanical
support and engenders a hydrophobic environment that is
important for conducting water and nutrients (Whetten and
Sederoff, 1995; Vanholme et al., 2010); furthermore, many
soluble phenolics, synthesized in a species-specific manner,
act as anti-pathogenic phytoalexins, antioxidants, or UVabsorbing compounds, protecting plants from biotic and
abiotic stresses, or act as pigments attracting pollinators, or
as signaling molecules mediating plant–microbe interactions
(Dixon and Paiva, 1995). The tissue-specific formation and
accumulation of phenylpropanoids probably represents one
of the most important prerequisites that ultimately facilitated
the colonization of land by early plants (Weng and Chapple,
2010).
The biosynthesis of phenylpropanoids entails a sequence of
central enzyme-regulated reactions (termed the general phenylpropanoid pathway), from which branch pathways emanate
toward different aromatic end products. In particular, this
metabolic pathway assimilates about 30–40% of the
biospheric organic carbon, ending up as the predominant
lignin polymer in the cell walls. The general phenylpropanoid
pathway in plants is linked to the shikimate pathway, which
produces aromatic amino acids from central carbon metabolisms. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is the
entry-point enzyme of the general phenylpropanoid pathway,
which channels L-Phe from the primary metabolic pool to
the synthesis of trans-cinnamic acid (t-CA). The t-CA produced is then further transformed into many phenolic compounds (Figure 1) (Bate et al., 1994; Cochrane et al., 2004).
PAL activity has been found in all the higher plants analyzed
so far, and in some fungi and a few bacteria, but not in
animals (Hodgins, 1971; Fritz et al., 1976; Xiang and Moore,
2005). Since its discovery in 1961 by Koukol and Conn
(1961), PAL has been studied extensively. Its activity is
stimulated in developmental programming and by a variety
of environmental cues, including pathogenic attacks, tissue
Published by the Molecular Plant Shanghai Editorial Office in association with
Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.
Molecular Plant 8, 17–27, January 2015 ª The Author 2015.
17
Nitrogen
Sugar
Suga
UV
Cytokinins
Molecular Plant
Regulation of Phenylalanine Ammonia-Lyase in Plants
basis for proteolytic regulation of this gateway enzyme is
highlighted.
Shikimate
ENZYME PROPERTIES AND PRODUCT
INHIBITION
MYB
SCF KFB/KMD
LIM
Phenylalanine
KNOX
PAL
B-type ARR
Cinnamate
Cytokinin
response
C4H
p-Coumarate
C3H
4CL
p-Coumaroyl CoA
HCT
Caffeic acid
Ferulic acid
Sinapic acid
Monolignols
CHS
Stilbenes
Lignin
Isoflavonoids
FLS
Anthocyanins
Condensed tannins
Flavonols
UGT78D1, 2
Flavonol glycosides
Figure 1. Schema of the Phenylpropanoid Biosynthesis
Pathway, Highlighting Molecular Regulation on the Gateway
Enzyme PAL and the Physical Interaction of the Early
Phenylpropanoid Pathway Enzymes.
The data are summarized primarily from studies in Arabidopsis, poplar,
and tobacco. The identified transcriptional regulation (MYB, LIM, KNOX
transcription factors), kelch repeat containing F-box protein-mediated
ubiquitylation and degradation, and metabolic feedback regulation on
PAL are indicated, respectively, in green, red, and blue. The dashed line
indicates negative regulation; the double arrow indicates multiple steps of
the pathway. 4CL, 4-coumarate-coenzyme A ligase; ARR, Arabidopsis
response regulator (to cytokinins); C4H, cinnamate 4-hydroxylase; CHS,
chalcone synthase; FLS, flavonol synthase; HCT, hydroxycinnamoylcoenzyme A:shikimate hydroxycinnamoyltransferase; UGT78D1,
flavonol 3-O-rhamnosyltransferase; UGT78D2, flavonoid 3-O-glucosyltransferase.
wounding, UV irradiation, exposure to heavy metals, low
temperatures, and low levels of nitrogen, phosphate, or ions
(Dixon and Paiva, 1995; Weisshaar and Jenkins, 1998). The
on and off, up and down activity of PAL in developmental
processes and in stress responses mirrors its sophisticated
regulatory control.
In the last few decades, studies in biochemistry, enzymology,
structural biology, and molecular genetics have suggested/unveiled multifaceted regulatory mechanisms that plant cells variously use to control the activity of PAL. The recognized control
of PAL can occur through several mechanisms, namely, product inhibition, transcriptional and translational regulation, posttranslational inactivation and proteolysis, enzyme organization/
subcellular compartmentation, and metabolite feedback regulation. In this review, an overview of the molecular bases
and mechanisms controlling the activity of PAL is presented.
In particular, recent progress in unraveling the molecular
18
PAL catalyzes the non-oxidative elimination of ammonia from
L-Phe to yield t-CA (Cochrane et al., 2004). This enzyme
belongs to a large amino acid ammonia-lyase/aminomutase superfamily, wherein it is phylogenetically clustered to the histidine
ammonia-lyases (HALs) and tyrosine ammonia-lyases (TALs)
found in bacteria (Poppe and Retey, 2005; Xiang and Moore,
2005). PAL from dicotyledonous plants predominantly and
efficiently deaminates L-Phe, while PAL from yeast and some
monocots, e.g., maize, converts L-Phe and L-tyrosine to their
corresponding cinnamic acid and p-coumaric acid (Fritz et al.,
1976; Ro¨sler et al., 1997).
Molecular Plant 8, 17–27, January 2015 ª The Author 2015.
In all plants studied so far, PAL is encoded by a multi-gene family, thus presenting as multiple isoforms (Cramer et al., 1989;
Wanner et al., 1995). PAL occurs as a tetrameric enzyme
in vivo with molecular weight of around 275–330 kDa (Havir
and Hanson, 1973; Zimmermann and Hahlbrock, 1975; Appert
et al., 1994). Multiple tetrameric forms with slightly different
molecular weights, isoelectric point values, and substrate
binding affinities have been purified from suspension cell
cultures of bean (Phaseolus vulgaris L.), suggesting that PAL
can form heterotetramers (Bolwell et al., 1985). When different
tobacco PAL isogenes are co-expressed in Escherichia coli,
the recombinant proteins can form heterotetramers (Reichert
et al., 2009).
Structurally, PAL is a helix-containing protein and its tertiary and
quaternary sizes are much larger than those of HALs; this is
mainly due to the existence in PAL of two additional structural
segments: a mobile N-terminal extension, which probably anchors the enzyme and interacts with other cellular components,
and a specific shielding domain sited over the active center,
which probably controls enzyme activity by restricting the access of the substrate to a narrow channel (Figure 2) (Calabrese
et al., 2004; Ritter and Schulz, 2004). PAL and other members
of the ammonia-lyase/aminomutase superfamily contain a common co-factor, 4-methyldiene-imidazol-5-one (MIO), in their
active site for catalysis (Figure 2); it is formed by the
autocatalytic cyclization and dehydration of an internal tripeptide segment, namely, Ala-Ser-Gly (Rother et al., 2002;
Alunni et al., 2003). MIO resides on top of the positive pole of
three polar helices in the active site and serves as a prosthetic
electrophile, arguably to initiate the direct elimination of the
a-amino group or for the stereospecific abstraction of the
b-proton to form aryl acid (Figure 2) (Langer et al., 2001;
Retey, 2003; Calabrese et al., 2004; Ritter and Schulz, 2004;
Louie et al., 2006).
PAL shows product inhibition properties; its activity is inhibited by
t-CA, the product of its catalyzed reaction (O’Neal and Keller,
1970; Appert et al., 1994). Although there are no detailed in vitro
studies of the inhibition mechanism, t-CA might act as a
competitive inhibitor (Camm and Towers, 1973; Sato et al.,
1982). Its derivatives, such as p-coumarate, O-chlorocinnamate,
Molecular Plant
Regulation of Phenylalanine Ammonia-Lyase in Plants
A
Figure 2. Comparison of the Tertiary
Structures of PAL Monomer from Parsley
(Petroselinum crispum) (A) and HAL from
Pseudomonas putida (B).
B
MIO
domain
MIO
domain
MIO
MIO
25(N)
Core
domain
Core
domain
(C)
Shielding
domain
and the related compounds p-hydroxybenzoate and
2-naphthoate, also can inhibit PAL activity (Sato et al., 1982). In
the crystal structure complex of TAL from Rhodobacter
sphaeroides, trans-cinnamate or p-coumarate was observed to
bind closely into the active site, implicating the structural basis
of ammonia-lyase for its product inhibition (Appert et al., 1994).
PAL purportedly follows an ordered Uni Bi–type reaction
sequence, in which trans-cinnamate is released before
ammonia from the active site (Williams and Hiroms, 1967).
Presumably, its release is a necessary step for inducing
conformational change in PAL, preparing it for the next catalytic
cycle. Therefore, a high concentration of trans-cinnamate may
impair the release of the enzymatic product and compete for the
binding of Phe.
Earlier studies showed that the purified PAL enzyme from parsley, French beans, or cultures of alfalfa cells shows negative
cooperativity; it has a high binding affinity (low Km) for phenylalanine at low concentration of substrate and a relatively low binding affinity (high Km) at a high substrate concentration
(Zimmermann and Hahlbrock, 1975; Bolwell et al., 1985; Jorrin
and Dixon, 1990). These observations suggest that the
substrate itself exerts an inhibitory effect on the rate of
product formation through negative cooperative regulation of
the enzyme subunits (Zimmermann and Hahlbrock, 1975).
However, later experiments revealed that heterologously
expressed recombinant PAL isoform in E. coli displays typical
(N)
The ribbon representations of PcPAL and PpHAL
are re-built based on the coordinates of PAL
(1W27) and HAL (1B8F) (Ritter and Schulz, 2004)
via the PyMol program. The polypeptide chains
are colored green for the MIO domain, magenta
for the helical bundle core domain, and cyan for
the inserted shielding domain (in PcPAL). The
first 24 residues (the extension domain) in the
crystal structure of PcPAL were missing (Ritter
and Schulz, 2004). The MIO is shown in the balland-stick model.
(C)
Michaelis-Menten kinetics and no cooperative behavior (Appert et al., 1994; Appert
et al., 2003). This mechanistic discrepancy
between the native and recombinant
enzymes was ascribed to the presence of
heterotetrameric isoforms in the purified
native enzymes or the occurrence of posttranslational modification, which may not
occur in the bacterial expression system.
However, the recombinant heterotetramer,
composed of tobacco PAL1 and PAL2 or
PAL1 and PAL4, also does not exhibit
negative cooperative kinetics (Reichert
et al., 2009). This suggests that the
cooperative regulation of PAL activity
observed in the earlier studies was either
an artifact or caused by unknown factors
rather than via the heteromerization of kinetically different
isoforms.
TRANSCRIPTIONAL REGULATION OF
THE EXPRESSION OF PAL GENES
PAL has long been known to be regulated at the transcriptional
level in response to environmental stimuli, and to developmental
cues, e.g., the demand for lignin in specific tissues (Edwards
et al., 1985; Liang et al., 1989; Dixon and Paiva, 1995; Anterola
et al., 2002; Pawlak-Sprada et al., 2011). In plants, PAL is
encoded by a multi-gene family, ranging from a few members
in many species, to more than a dozen copies in potato and tomato (Chang et al., 2008). Specific PAL isogenes often exhibit a
unique but overlapping spatial and temporal expression
pattern, suggesting that PAL isoforms might have distinct but
redundant functions in plant growth, development, and in
plant–environment interactions. For instance, in Populus spp.,
two PAL cDNAs are expressed differentially in developing shoot
and root tissues. PtPAL1 is mainly expressed in the cells of
non-lignifying stems, leaves, and roots, wherein cells accumulate
a high level of condensed tannins, whereas PtPAL2 is expressed
both in the heavily lignified structural cells of shoots and in the
non-lignifying cells of root tips, highlighting the potential spatial
association of PAL isoforms with specific metabolic activities
(Kao et al., 2002). In Arabidopsis, four PAL isogenes are
expressed in inflorescence stems, a tissue rich in lignifying
Molecular Plant 8, 17–27, January 2015 ª The Author 2015.
19
Molecular Plant
Regulation of Phenylalanine Ammonia-Lyase in Plants
cells; AtPAL-1, -2, and -4 have much higher expression levels
than AtPAL-3 (Wanner et al., 1995; Raes et al., 2003; Rohde
et al., 2004). Genetic evidence from studies of mutant lines
deficient in AtPAL isogenes has established the close
association of AtPAL1, 2, and 4 with lignin biosynthesis (Oh
et al., 2003; Rohde et al., 2004; Huang et al., 2010). Meanwhile,
it is found that AtPAL1 and PAL2 also dominate flavonoid
synthesis; the pal1 pal2 double knockout mutant is almost
devoid of flavonoids, although PAL activity reaches 50% of the
level of the wild type due to the activity of other isoforms
(Rohde et al., 2004). Nitrogen depletion particularly induces the
expression of PAL1 and PAL2; together with the presumable
induction of the downstream phenylpropanoid–flavonoid
biosynthetic genes, it increases the accumulation of flavonoids
(kaempferol and quercetin), anthocyanins, and sinapic acid
(Olsen et al., 2008). These data indicate that AtPAL isogenes
differentially respond to environmental stresses and that
AtPAL1 and AtPAL2 have functional specialization in
environmentally triggered phenolic synthesis.
expression of PAL (Martin and Paz-Ares, 1997). AmMYB305,
which is expressed in the carpels of young snapdragon flowers
(Antirrhinum majus) activates the PAL promoter when it is coexpressed in tobacco protoplasts (Sablowski et al., 1994).
In the anthers of tobacco, a specific transcription factor,
NtMYBAS1/2, can bind to the MYB binding site and activate
NtPAL1 expression, thus positively regulating the expression of
NtPAL1 and phenylpropanoid synthesis in sporophytic tissues,
such as the tapetum, stomium, and the vascular tissues of
young tobacco anthers (Yang et al., 2001). In Arabidopsis, a
hierarchical regulatory network controls the formation of
secondary cell walls, in which three MYB transcription factors,
MYB58, MYB63, and MYB85, are specifically involved in
regulating lignin biosynthesis (Zhou et al., 2009). Mutation of the
AC elements abolishes the binding of MYB58. It is unknown
whether, and how, these transcription factors can directly
target the AtPAL promoter and control the temporal and spatial
expression of AtPALs in coordinating the distribution of carbon
flux to different cell-wall polymers. In addition to the MYB transcription factors, the LIM domain-containing proteins, which
are composed of a double, semi-conservative zinc finger motif,
have also been shown to bind on the AC-rich motif, the Pal-box
[CCA(C/A)(A/T)A(A/C)C(C/T)CC], and positively regulate the
expression of NtPAL and other phenylpropanoid genes
(Kawaoka et al., 2000; Kawaoka and Ebinuma, 2001).
Furthermore, the KNOX family of transcription factors, which
function in maintaining cells in an indeterminate state (Tsiantis,
2001), also affect PAL expression. Mutations in the KNOX gene
BREVIPEDICELLUS (BP) upregulate the expression of
Arabidopsis PAL1, 4CL1, C4H, and CAD1 and cause premature
lignification of the vasculature with the phenotype of shorter
internodes, downward-pointing siliques, and an epidermal stripe
of disorganized cells along the stem, all of which suggest that the
KNOX transcription factor AtBP functions as a negative regulator
for the expression of AtPAL and other lignin biosynthetic genes
(Figure 1) (Mele et al., 2003). Again, it will be interesting to
determine whether this family of transcription factors directly or
indirectly targets PAL and other phenylpropanoid biosynthetic
genes, and how they coordinately control cell-wall lignification
with other regulators in the network.
The developmental and inducible expression of PALs is regulated
by the functioning of their promoter activity. Both AtPAL1 and
AtPAL2 promoter-GUS activities are readily detected throughout
the lifetime of a plant, from the early seedlings to fully grown adult
plants, in which they are strongly expressed in the vascular tissues of roots, leaves, and inflorescence stems, but not active in
young and fast-growing tissues such as the root tip or the shoot
apical meristem (Ohl et al., 1990; Wong et al., 2012).
Mapping the promoter region of AtPAL1 via various deletions reveals that the 1.8-kb promoter possesses a possible negative
element between 1832 and 816, where the deletion increases
the promoter activity by 30%; two potential positive elements are
located thereafter between 816 and 290, where the deletion
severely lowers the activity of the promoter to about 2% of that
of the full-length one (Ohl et al., 1990). This study revealed that
the proximal region of the promoter to location 290 sustains the
tissue- and organ-specific expression pattern as well as a fulllength promoter does; furthermore, the proximal region to 540
is responsive to environmental stimuli (Ohl et al., 1990). These
discoveries can help to further identify the particular transcription
factors that interact with these promoter regions for controlling
tissue-specific and stimuli-inducible expression of PAL.
The dissected promoter regions typically contain multiple cisregulatory elements (Yang et al., 2001; Rohde et al., 2004). In
particular, the AC-rich motifs (AC-I, ACCTACC; AC-II, ACCAACC;
and AC-III, ACCTAAC), the target binding sites of the MYB transcription factors, commonly occur in the promoters of PAL isogenes from different species. These AC cis-elements also exist
in the promoters of many other phenylpropanoid biosynthetic
genes, including the chalcone synthase (CHS) gene, and most
of the monolignol biosynthetic genes, such as 4CL (Lois et al.,
1989; Hatton et al., 1995; Hatton et al., 1996; Zhong and Ye,
2009; Zhao and Dixon, 2011), highlighting a key role of such
MYB binding elements in coordinating the expression of a set
of phenylpropanoid biosynthetic genes for the tissue-specific
synthesis of phenolics (Leyva et al., 1992).
A few R2R3 MYB transcription factors are known to be able to
trans-activate PAL promoters to control the tissue-specific
20
Molecular Plant 8, 17–27, January 2015 ª The Author 2015.
POST-TRANSLATIONAL
MODIFICATIONS OF PAL
Ubiquitination and Proteolysis of PAL
Under biotic and abiotic stresses, PAL activity is induced as a
defense-response mechanism, concomitant with the onset of
the biosynthesis of different phenolics (Dixon and Paiva, 1995).
As demonstrated for the PAL of French bean, its induction
essentially reflects de novo enzyme synthesis after the
upregulation of messenger RNA levels (Edwards et al., 1985).
However, after the peak of the activity, PAL rapidly declines in
activity, engendering a transient induction pattern. This
behavior suggests the imposition of a post-translational regulation (Lamb et al., 1979; Jones, 1984). Early studies proposed an
inactivation mechanism that reversibly converts PAL into its
inactive form: The elicitation triggers the synthesis of a putative
proteinaceous inhibitor that can react specifically with the PAL
enzyme, thus engendering an inactive enzyme–inhibitor
complex (Attridge and Smith, 1974; French and Smith, 1975).
Regulation of Phenylalanine Ammonia-Lyase in Plants
Molecular Plant
Purportedly, a high-molecular-weight protein fraction from sunflower leaves possesses a PAL inactivation system (Creasy,
1976). However, this has not been confirmed or characterized.
In examining the fluctuation of PAL activity in the injured root
tissue of sweet potato, Tanaka and Uritani (1977) reported that
after the de novo synthesis of PAL, its subsequent decline in
activity likely reflects the proteolytic degradation of the existing
enzyme; however, its molecular basis was not resolved until
recently.
(Zhang et al., 2013). These data denote that the KFBs identified
are negative regulators governing the stability of PAL via the
ubquitination–26S proteasome pathway.
In eukaryotic systems, the ubiquitin-26S proteasome system
dominates selective protein degradation (Vierstra, 2003). To
initiate its breakdown, the protein is first modified by the
covalent attachment of the ubiquitin, a 76 amino acid protein,
to its lysine residues. Then, the protein is recognized and
degraded by the 26S proteasome (Smalle and Vierstra, 2004).
Using ubiquitin affinity and nickel-chelate affinity chromatography coupled with mass spectrometry, an array of ubiquitinated
proteins from Arabidopsis was identified (Saracco et al., 2009;
Kim et al., 2013a). Among them, several phenylpropanoidmonolignol biosynthetic enzymes, including AtPAL-1, -2, and
-3, were found to be potentially ubiquitinated (Kim et al.,
2013a). The (poly)ubiquitination of four Arabidopsis PAL
isoforms was confirmed in our recent immunoblot analysis of
transiently expressed AtPAL proteins against the anti-ubiquitin
antibody (Zhang et al., 2013).
Ubiquitination of a target protein requires the sequential action of
three enzymes: ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin protein ligase (E3) (del Pozo and
Estelle, 2000; Lechner et al., 2006). When Arabidopsis PAL1 and
PAL2 are used as the bait to screen an Arabidopsis cDNA
expression library in a yeast two-hybrid assay, three interacting
partners are captured, denoted kelch motif-containing F-box
proteins, KFB0-1, -20, and -50. Subsequent pair-wise verification
via Y2H, BiFC, and co-IP assays corroborates the physical interactions of all four AtPAL isozymes with these KFB proteins
(Zhang et al., 2013). Furthermore, a close homolog, KFB39, also
interacts with AtPALs (Zhang et al., 2014). This finding agrees
well with the discovery in a genome-wide study of binary protein–protein interactions that AtPAL1, 2, and/or 4 are found to
potentially interact with KFB01 and KFB50 (Arabidopsis
Interactome Mapping Consortium, 2011).
The F-box proteins are the structural components of the canonical SCF type E3 protein–ubiquitin ligase, responsible for specifying the target proteins for ubiquitination and degradation (del
Pozo and Estelle, 2000; Lechner et al., 2006). The KFB proteins
are one subclass of F-box proteins. The physical interaction of
these KFBs with PAL isozymes suggests that they might act as
key mediators specifying the ubiquitination of PAL and
subsequent degradation. Indeed, co-expressing any one of
them with PAL isozyme in tobacco leaves impairs the stability
and activity of the latter. Moreover, with treatment of MG132, a
specific inhibitor of the 26S proteasome, the degradation of
PAL protein is retarded or mitigated; meanwhile, the ubiquitination of PAL is enhanced. Furthermore, manipulating (either upregulating or downregulating) KFB expression in Arabidopsis reciprocally affects the cellular concentration and activity of PAL and
the consequent accumulation of phenolic esters, flavonoids, anthocyanins, and the lignin deposited in the cell walls of the stem
E3 ligase–mediated ubiquitination and the subsequent protein
degradation are critical components in the complex regulatory
network underlying cellular processes (Vierstra, 2003). In most
cases, the E3 ligase components themselves are regulated by
the developmental cues of the plant and by environmental
stresses, thus transducing developmental or environmental
signals to the corresponding physiological processes (del Pozo
and Estelle, 2000; Lechner et al., 2006). Examining the
expression of the KFBs identified reveals that they differ in
tissues and at different growth stages; for example, KFB20 is
highly expressed in leaf and stem tissues, while KFB01 is
mainly expressed in flowers, thus denoting their potential
distinct roles in mediating PAL degradation and controlling a
particular set of phenylpropanoid synthesis. Moreover, the
expression of four KFBs is affected by stimulators. Exposing
Arabidopsis seedlings to a high source of carbon (4% sucrose
in the medium) or to UVB irradiation substantially suppresses
the expression of KFB and is accompanied by an increased
accumulation of flavonoids/anthocyanin pigments (Zhang et al.,
2013).
The tissue-specific, temporal, and inducible expression of the
KFB genes identified and the reciprocal correlation of their
expression with the cellular concentration of PAL and the accumulation of phenolics strongly suggest that KFB-mediated PAL
ubiquitination and degradation function as a basic regulatory
mechanism, by which plant cells finely gear carbon flux toward
phenylpropanoid metabolites.
The close homologs of the Arabidopsis KFB genes identified can
be recognized across a range of plants, from moss (Physcomitrella patens) to angiosperm and gymnosperm trees (e.g., Populus and Picea). All those species synthesize and accumulate
different types of phenylpropanoids (Richardson et al., 2000;
Morreel et al., 2004; Richter et al., 2012). Although the precise
functions of those KFB homologs remain unknown, the
evolution of those E3 ligase subunits in land plants, along with
the emergence of phenolic metabolites, implies that the KFBmediated selective turnover of PAL may be a universal regulatory
mechanism adopted by a variety of terrestrial plants to control
their phenylpropanoid synthesis.
Besides mediating PAL turnover, the same set of KFBs was found
to physically interact with several type B Arabidopsis response
regulators; accordingly, these KFBs are proposed to negatively
regulate cytokinin responses (Kim et al., 2013b). Cytokinins are
a group of plant hormones with vital roles in regulating cellular
division and modulating the activities of both the shoot and root
meristems in building plant architectures (Sakakibara, 2006).
Several studies suggested that disturbing the translocation,
distribution, or signal perception of cytokinin in plants severely
degrades their vascular formation and lignification (Werner
et al., 2003; Zhang et al., 2014), inferring the existence of
common links orchestrating the cytokinin signaling responses,
xylogenesis, and lignin deposition. Further research will clarify
whether the KFB proteins identified have potential roles in
coordinating cytokinin signaling and phenylpropanoid–lignin
Molecular Plant 8, 17–27, January 2015 ª The Author 2015.
21
Molecular Plant
Regulation of Phenylalanine Ammonia-Lyase in Plants
biosynthesis and/or deposition. It would also be interesting to
systematically identify any other potential targets of those KFBs
and to explore whether the altered plant lignification in turn
affects other KFB-mediated biological processes. Since the
expression patterns of four KFB genes are different (Zhang
et al., 2013), defining and delineating their potential different
biological functions in planta would be valuable.
1986). Both PAL activity and PAL gene transcription are
negatively regulated by t-CA, either via the exogenous
application of the compound or by blocking the downstream
C4H activity for metabolizing cinnamic acid (Lamb, 1979;
Bolwell et al., 1986; Mavandad et al., 1990; Blount et al., 2000).
When PAL from French bean is overexpressed in tobacco,
wherein the C4H is co-suppressed, the transgenic plants present
significantly lower PAL activity than those plants harboring the
bean PAL alone, suggesting that PAL activity might be feedback
regulated by the accumulated trans-cinnamate due to the reduction of C4H in planta (Blount et al., 2000). The mechanism by
which PAL is feedback downregulated may be complex.
Excessive trans-cinnamate can directly inhibit the enzyme
activity itself, since PAL exhibits a product inhibition property,
or the accumulated trans-cinnamate impairs PAL transcription
(Lamb, 1979; Bolwell et al., 1986; Mavandad et al., 1990). In
addition, our current study also suggests that feeding transcinnamate to Arabidopsis seedlings induces the expression of
the identified KFB proteins that modulate PAL turnover and the
pathway activity (Zhang, X., and Liu, C.J., unpublished results).
Phosphorylation of PAL
Phosphorylation is another major type of post-translational modification of proteins in eukaryotic cellular processes. Bolwell
(1992) reported the detection of PAL phosphorylation in suspension cultures of French bean cells (Bolwell, 1992). Their
subsequent purification led to the identification of a 55-kDa
PAL kinase activity that phosphorylates the threonine/serine residues of the conserved polypeptides of the PALs of both bean
and poplar; such PAL kinase displays the property of a
calmodulin-like domain protein kinase (CDPK) (Allwood et al.,
1999). In assaying eight protein kinases from Arabidopsis, a
calcium-dependent protein kinase, AtCDPK, could phosphorylate the short polypeptide of PAL and a poplar recombinant
PAL enzyme (Cheng et al., 2001). The sequence of the
polypeptide of PAL used in the assay is largely conserved in
homologous enzymes from other species; therefore, this finding
implies that this threonine/serine type of phosphorylation of
PAL might be a ubiquitous modification mechanism occurring
in higher plants (Allwood et al., 1999). Structural determination
of PAL from parsley suggests that the potential phosphorylation
site of PAL might be located at the end of a flexible helix
connecting the shielding domain to the core domain. This
position displays the highest mobility, suggesting that
phosphorylation might affect the shift of the shielding domain
and change access to the active center of the enzyme (Ritter
and Schulz, 2004). In addition, PAL phosphorylation was also
predicted to mark particular subunits for turnover or to target
them to the membrane for subcellular compartmentalization
(Cheng et al., 2001). However, recent phosphoproteomic
studies failed to confirm the phosphorylation of PAL and any
other monolignol proteins from poplar (P. trichocarpa). In one
study, 147 phosphoprotein groups were identified from
differentiated xylem, wherein monolignol biosynthetic enzymes
are abundant. None of them are proteins known to participate
in the monolignol biosynthetic pathway (Chen et al., 2013). In
another work, Liu et al. (2011) detected 151 phosphoproteins in
the dormant terminal buds of P. simonii 3 P. nigra using the
same strategy of phosphopeptide enrichment; similarly, none
are monolignol proteins (Liu et al., 2011). Further study may
clarify whether the apparent disparity between the early and
current studies reflects the limitations of the methodology used,
and whether phosphorylation of PAL and/or other monolignol
enzymes is a species-specific or a condition-specific event.
METABOLITE FEEDBACK REGULATION
Apart from transcriptional regulation in response to environmental stimulus and nutrient balance, PAL and phenylpropanoid
biosynthetic activity appear to be metabolically regulated by
particular biosynthetic intermediates or chemical signals
(Figure 1). t-CA has long been proposed as a signal molecule,
regulating flux into the pathway (Lamb, 1979; Bolwell et al.,
22
Molecular Plant 8, 17–27, January 2015 ª The Author 2015.
The feedback regulation is not only triggered by the immediate
product of PAL but also by the intermediates from branch pathways or exogenous chemical signals (Figure 1). Feeding caffeic
acid to soybean (Glycine max) seedlings substantially inhibits
PAL activity (Bubna et al., 2011). In the Arabidopsis double
mutant deficient in AtUGT78D1 and UGT78D2, which
compromises the 3-O-glycosylation of flavonols, AtPAL activity
and the transcripts of both AtPAL1 and PAL2, the two
isogenes specified for flavonoid synthesis (Olsen et al., 2008),
and CHS are inhibited, so repressing flavonol synthesis itself.
Moreover, upon blocking flavonol synthesis either in the tt4/chs
or flavonol synthase 1 (fls1) mutant lines, the repression of PAL
can be released completely, suggesting that flavonol aglycones
might serve as signal molecule triggering PAL feedback
regulation (Figure 1) (Yin et al., 2012). The ugt78d1 ugt78d2–
dependent suppression of the flavonol branch and AtPAL1 and
PAL2 activity does not affect the synthesis of other
phenylpropanoids; in particular, lignin biosynthesis appears
normal, corroborating the functional specification of AtPAL1
and PAL2 in flavonoid synthesis.
MACROMOLECULAR ORGANIZATION
OF PAL WITH OTHER
PHENYLPROPANOID ENZYMES
Srere in 1985 proposed the term ‘‘metabolons’’, referring to the
super-complex formed by the interactions of sequential metabolic enzymes and their interaction with supporting cellular structural elements (Srere, 1985). Such organization of metabolic
pathways is believed to increase the local concentrations of the
enzymes and of their substrates, and to improve intermediate
handing over between consecutive enzymes. In addition, it also
defines some specific domains in the spaces of cell to ensure
the swift conversion of the labile or toxic intermediates and
avoids unnecessary competition with other metabolites present
in the cell (Srere, 1987, 2000; Jorgensen et al., 2005). In
eukaryotes, most of the cytochrome P450s are anchored via
their N terminus membrane-targeting peptides on the cytoplasmic surface of the endoplasmic reticulum (ER), with their
Regulation of Phenylalanine Ammonia-Lyase in Plants
Molecular Plant
main catalytic domains protruding on the surface of the membrane (Bayburt and Sligar, 2002). This unique character of the
P450 enzymes provides perfect nucleation sites for forming
metabolons, namely, a gathering of some of the soluble
enzymes, thus increasing metabolic efficiency. The
phenylpropanoid pathway has long been proposed to have such
macromolecular organization (Winkel-Shirley, 1999; Winkel,
2004; Jorgensen et al., 2005). By differential centrifugation,
sedimentation of microsomes, or gradient separation of different
membrane fractions, PAL activity from different plant species is
found to associate with the ER membrane (Czichi and Kindl,
1975, 1977; Hrazdina and Wagner, 1985). The membrane
fraction from potato tubers converts Phe more efficiently into
p-coumaric acid than does exogenously supplied cinnamic acid.
This finding engenders an interesting assumption, i.e., the
potential association of PAL with the consecutive P450 enzyme,
C4H, for metabolite channeling. Using isotope-labeled Phe and
cinnamic acid, Rasmussen and Dixon (1999) showed that the
exogenous application of cinnamate into suspension cultures of
tobacco cells does not dilute/equilibrate with the endogenous
pool of cinnamic acid produced from PAL, supporting the
concept of metabolic channeling between PAL and C4H. This
proposed concept was further corroborated in subsequent
studies of the co-localization of tobacco PAL and C4H (Achnine
et al., 2004). Immunoblot analysis on the prepared microsomal
fractions and immunofluorescence confocal microscopy
observations reveal that the operationally soluble tobacco PAL1
itself partially bounds to the ER membrane and co-localizes with
NtC4H, whereas its isozyme, NtPAL2, appears mostly as a cytosolic protein. However, when co-overexpressed with C4H, both
isoforms shift to the membrane fraction. These data suggest
that membrane-bound C4H might serve as the nucleation site
for binding the PAL isoforms and that NtPAL1 binds more strongly
to C4H than NtPAL2 does (Achnine et al., 2004).
soluble enzyme, 4-coumarate: coenzyme A ligase (4CL), is also
redistributed closer to the ER. Their data suggest that AtC4H,
AtC3H, and two functionally connecting soluble enzymes,
At4CL and AtHCT, likely form a loosely associated supramolecular complex in the cells. Moreover, when plant cells are under
wounding stress that induces cell-wall lignification and the
defensive production of phenolics, such protein association
is enhanced, suggesting the formation of metabolon appropriate
for particular metabolic needs (Bassard et al., 2013). These
experimental data substantiate the concept that phenylpropanoid
biosynthetic enzymes, including different PAL isozymes, are
nucleated by membrane-bound P450 proteins; in this manner,
they form enzyme complexes to regulate pathway activity and
metabolic complexity.
The scope of metabolon sitting on the general phenylpropanoid
pathway to the monolignol biosynthetic branch was expanded
recently. Using different biochemical approaches, Chen et al.
(2011) demonstrated the interaction between two poplar C4H
isoforms and their interactions with C3H. Those isoforms form
heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/
C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane–
protein complexes. Moreover, the authors found that the
assembly of those hydroxylase isoforms significantly improves
the enzyme kinetics with any of the complexes by some 70–
6500 times higher than that of the individual proteins.
Furthermore, the association of PtC4H and PtC3H likely creates
two potential hydroxylation pathways for monolignol synthesis:
one through p-coumaric acid to caffeic acid and the other,
through p-coumaroyl shikimic acid to caffeoyl shikimic acid.
By protein co-purification and fluorescence resonance energy
transfer studies, Bassard et al. (2013) reported that Arabidopsis
C4H and C3H, like their homologs in poplar, also interact with
each other to form homomers or heteromers and both are
mobile within the ER. Furthermore, AtHCT, the first branchpoint enzyme for monolignol synthesis and an operationally soluble protein, was revealed to partially associate with the ER, and
the expression of AtC3H in the cells promotes the association
of hydroxycinnamoyl-coenzyme A:shikimate hydroxycinnamoyltransferase with the ER. Under AtC3H overexpression, another
CONCLUSIONS AND PERSPECTIVES
The gateway enzyme, PAL, plays a key role in mediating carbon
flux from primary metabolism into the phenylpropanoid pathway.
Over the decades, studies have shown that exquisite regulatory
mechanisms at multiple levels control the transcription and the
enzymatic activity of PALs, thereby coordinating the distribution
of metabolic flux into a set of aromatic natural products and the
abundant biopolymer, lignin.
At the transcriptional level, the PAL promoter contains characteristic cis-regulatory elements, which recruit particular transcription factors to regulate PAL developmental and inducible
gene expression. Recognizing the ubquitination of PAL and identifying a group of ketch-repeat F-box proteins that mediate PAL
proteolytic degradation has shed light on our understanding of
the post-translational regulation of phenylpropanoid biosynthesis and of the cross talk of phenolic metabolisms with other
biological processes. Moreover, there is increasing evidence
pointing to the organization of phenylpropanoid pathway enzymes. More detailed studies are needed to gain a comprehensive insight into the complexity of the molecular regulation of
PAL activity and the related phenylpropanoid biosynthesis. For
instance,
1) Although a few transcription factors are implicated in the
control of the PAL gene expression, a detailed characterization remains to be conducted to define their interactions
with cis-regulatory elements of PAL and their regulatory
roles in modulating specific phenylpropanoid synthesis in
response to development and environmental clues.
2) PAL isozymes form homotetramers or heterotetramers.
Both Y2H and BiFC assays indicate that the AtKFB proteins
identified likely interact differentially with AtPAL isozymes.
It will be interesting to determine whether the homotetramerization or heterotetramerization of PALs and their differential interactions with KFB proteins have any significance in regulating the pathway activity.
3) The AtKFB genes identified exhibit tissue-specific and
developmental expression patterns. They also respond
differentially to environmental stimuli. The differential regulatory roles of KFBs in balancing carbon and nutrient sources for building plant architecture, cell-wall lignification,
and defense responses remain to be evaluated.
4) Besides ubiquitination, it remains unclear whether PALs undergo any other types of post-translational modifications;
Molecular Plant 8, 17–27, January 2015 ª The Author 2015.
23
Molecular Plant
particularly, we need to re-examine whether phosphorylation modification occurs on PAL and/or other monolignol
enzymes and whether this type of modification is a
species-specific or a condition-specific event.
5) PAL is feedback regulated by its own product (at least in
the C4H– disruption plants), or by the intermediate of the
downstream branch pathway. More studies of genetics,
biochemistry, and structure biology are needed to detail
the molecular mechanisms underlying such feedback
regulation and to elucidate whether the metabolites have
a direct or an indirect effect on PAL activity.
Phenylpropanoid biosynthesis controls up to 40% of organic
compounds circulating in the biosphere. They contribute to the
hardiness, color, taste, and odor of plants. Many are of high economic value. Knowledge about the complicated regulatory mechanisms of the phenylpropanoid pathway, in particular the entrypoint enzyme PAL, will facilitate our sophisticated manipulation
of this economically important metabolic pathway toward the
desired agro-industrial and horticultural traits.
FUNDING
This material is based upon work supported by the U.S. Department of
Energy, Office of Science, Office of Basic Energy Sciences, Chemical
Sciences, Geosciences, and Biosciences Division under contract number
DEAC0298CH10886 and the National Science Foundation through grant
MCB-1051675 to CJL.
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No conflict of interest declared.
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Received: March 31, 2014
Accepted: October 25, 2014
Published: November 9, 2014
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