Biochem. J. (2012) 444, 11–25 (Printed in Great Britain)
11
doi:10.1042/BJ20120245
REVIEW ARTICLE
Gibberellin biosynthesis and its regulation
Peter HEDDEN1 and Stephen G. THOMAS
Rothamsted Research, Harpenden AL5 2JQ, U.K.
The GAs (gibberellins) comprise a large group of diterpenoid
carboxylic acids that are ubiquitous in higher plants, in which
certain members function as endogenous growth regulators,
promoting organ expansion and developmental changes. These
compounds are also produced by some species of lower plants,
fungi and bacteria, although, in contrast to higher plants, the
function of GAs in these organisms has only recently been
investigated and is still unclear. In higher plants, GAs are
synthesized by the action of terpene cyclases, cytochrome P450
mono-oxygenases and 2-oxoglutarate-dependent dioxygenases
localized, respectively, in plastids, the endomembrane system
and the cytosol. The concentration of biologically active GAs
at their sites of action is tightly regulated and is moderated
by numerous developmental and environmental cues. Recent
research has focused on regulatory mechanisms, acting primarily
on expression of the genes that encode the dioxygenases involved
in biosynthesis and deactivation. The present review discusses
the current state of knowledge on GA metabolism with particular
emphasis on regulation, including the complex mechanisms for
the maintenance of GA homoeostasis.
INTRODUCTION
biosynthetic precursors and metabolites, which are often present
at much higher concentrations than the hormones themselves.
Gibberellin A3 , also known as gibberellic acid, is best known
as the main GA product of G. fujikuroi (now reclassified as
Fusarium fujikuroi), from which it is produced for commercial
application, but it is also endogenous in some higher plant species.
Developing seeds often contain very high concentrations of GAs,
as well as a broad range of different structural forms. In fact, the
vast majority of the different GA structures were identified from
immature seeds, whereas vegetative and early reproductive tissues
contain a relatively restricted and conserved array of structures,
representing members of the biosynthetic pathways to GA1 and
GA4 and their metabolites. Although there is clear evidence for
a regulatory role for GAs in early seed development [2], the
function of the very high concentrations of active and inactive
GAs present in endosperm, cotyledons and/or testa of seeds at later
developmental stages is unclear. Seeds approaching maturity often
contain high levels of GA-inactivating activity, ensuring against
concentrations of bioactive GAs in mature seed that could provoke
premature germination and abnormal seedling growth [3].
Physiologically, a major function of GAs in higher plants can be
generalized as stimulating organ growth through enhancement of
cell elongation and, in some cases, cell division. In addition, GAs
promote certain developmental switches, such as between seed
dormancy and germination, juvenile and adult growth phases,
and vegetative and reproductive development. In this last case,
GAs may promote the vegetative or reproductive state, depending
The name GA (gibberellin) encompasses a large group of
tetracyclic diterpenoid carboxylic acids with the ent-gibberellane
(C20 ) or 20-nor-ent-gibberellane (C19 ) carbon skeletons, as
exemplified by the simplest members of each class, GA12
and GA9 respectively (Figure 1). Although first identified as
secondary metabolites of the fungus Gibberella fujikuroi, they
are ubiquitous in higher plants, in which certain members of
the group function as endogenous growth regulators. They are
also present in certain species of endophytic bacteria, several
fungal species and some lower plants, but their function in
these organisms is unclear. Currently 136 gibberellin structures
have been identified from all sources and are assigned the
trivial names gibberellin A1 –A136 , abbreviated to GA1 etc.,
numbered in order of discovery [1]. Their structures can be
found at http://www.plant-hormones.info/gibberellins.htm. There
are certainly further GAs still to be discovered, but due to their
very low abundance, their chemical characterization is far from
trivial, depending on comparison with chemically synthesized
standards, so that identification of new GAs has become an
increasingly rare event.
Relatively few GAs have intrinsic biological activity in higher
plants, the most common active forms being GA1 , GA3 and
GA4 (Figure 1). The universal occurrence of GA1 and GA4 in
plants suggests that these are the functionally active (hormonal)
forms, at least for growth promotion; they co-occur with their
Key words: gibberellin biosynthesis, gibberellin deactivation,
gibberellin homoeostasis, plant development.
Abbreviations used: ABA, abscisic acid; AG, AGAMOUS; AGF1, AT-hook protein of GA feedback 1; bHLH, basic helix–loop–helix; bZIP, basic leucine
zipper; CBF, C-repeat-binding factor; CHO1, CHOTTO1; CPS, ent -copalyl diphosphate synthase; DAG1, DOF AFFECTING GERMINATION 1; DDF, DWARF
AND DELAYED FLOWERING 1; DIF, difference between day temperature and night temperature; DOG1, DELAY OF GERMINATION 1; DRE, dehydrationresponse element; DREB, DRE-binding; EUI, ELONGATED UPPERMOST INTERNODE; FUS3, FUSCA3; GA, gibberellin; GA2ox, GA 2-oxidase; GA3ox, GA
3-oxidase; GA20ox, GA 20-oxidase; GAMT, GA methyl transferase; GGPP, trans -geranylgeranyl diphosphate; GGPS, trans -geranylgeranyl diphosphate
synthase; GID, GIBBERELLIN-INSENSITIVE DWARF; GUS, β-glucuronidase; HY5, ELONGATED HYPOCOTYL 5; JAZ, JASMONATE ZIM-domain protein;
KAO, ent-kaurenoic acid oxidase; KNOX, KNOTTED-like homeobox; KO, ent -kaurene oxidase; KS, ent -kaurene synthase; MADS, MCM1 (minichromosome
maintenance1), AGAMOUS, DEFICIENS and SRF (serum-response factor); LD, long day; LEC, LEAFY COTYLEDON; MS, male-sterile; ODD, 2-oxoglutaratedependent dioxygenase; P450, cytochrome P450 mono-oxygenase; PCD, programmed cell death; PIF, PHYTOCHROME-INTERACTING FACTOR; PIL5,
PIF3-LIKE 5; RGA, REPRESSOR OF ga1 (DELLA protein); RGL3, RGA-LIKE PROTEIN 3; RSG, REPRESSION OF SHOOT GROWTH; SCL3, SCARECROWLIKE 3; SD, short day; SLR1, SLENDER1; SOM, SOMNUS; SPT, SPATULA; T-DNA, transferred DNA; YAB1, YABBY1.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
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P. Hedden and S. G. Thomas
THE GA BIOSYNTHETIC PATHWAY
Figure 1
Structures of selected GAs
Included are the simplest example of a C20 -GA, GA12 , with the carbon numbering system, and
the simplest C19 -GA, GA9 . The principal biologically active GAs in higher plants are also shown:
GA1 , GA3 and GA4 .
on species. Gibberellins have an important role in fertility, as, in
addition to allowing stamen elongation, they are necessary for the
development, release and germination of pollen and for pollen
tube growth. In carrying out their functions GAs act in response
to developmental and environmental cues, which may regulate
GA biosynthesis, turnover (deactivation), perception or signal
transduction, sometimes acting at several points in the pathway.
Impressive progress has been made within the last decade in
establishing details of the GA biosynthesis and signal transduction
pathways. The advances in our understanding of the GA
signal transduction cascade (reviewed in [4–7]) have stemmed
particularly from the discovery that GID (GIBBERELLININSENSITIVE DWARF) 1 encodes a soluble GA receptor in rice
[8]. A pathway has emerged in which growth repression is exerted
by DELLA proteins through their regulation of transcription
([9]; also reviewed in [4]). In response to environmental and
developmental stimuli, this repression is relieved through the
production of biologically active GAs, which rapidly promote
DELLA degradation. Gibberellins bind within a pocket of GID1,
causing a conformational change that facilitates the formation of
a GA–GID1–DELLA complex and the subsequent ubiquitination
of DELLAs and their destruction by the 26S proteasome ([10–12];
also reviewed in [7]). In the absence of a functional degradation
pathway, binding of GA–GID1 has also been demonstrated to have
a repressive effect on DELLA function, although the physiological
significance has yet to be established [13,14]. There is currently
little evidence that DELLAs act as transcription factors that
directly control the expression of GA response genes. However,
recent studies demonstrated that DELLA repression of hypocotyl
expansion is mediated through their binding and sequestration
of the PIF (PHYTOCHROME-INTERACTING FACTOR) 3 and
PIF4 bHLH (basic helix–loop–helix) transcription factors [15,16].
There is now strong evidence that DELLAs also regulate the
activity of other classes of transcriptional regulators, including
the JAZs (JASMONATE ZIM-domain proteins). In this case,
DELLAs were shown to modulate jasmonate signalling by
binding to JAZs and thereby blocking their inhibitory effect on
downstream transcriptional events [17].
The present review focuses on GA biosynthesis and
deactivation: after a discussion of the pathways, we will highlight
recent advances in our understanding of how they are regulated.
c The Authors Journal compilation c 2012 Biochemical Society
This topic was reviewed previously by Yamaguchi [18]. In higher
plants, GAs are formed primarily from the methylerythritol
phosphate pathway [19], by which the hydrocarbon intermediate
ent-kaurene is produced from GGPP (trans-geranylgeranyl
diphosphate) in proplastids [20]. The biosynthetic pathway from
GGPP to the bioactive GAs is presented in Figure 2. It has been
generally assumed that ent-kaurene synthesis competes with that
of other GGPP-derived metabolites for a common pool of GGPP
(e.g. [21]). However, the finding that loss of geranyl diphosphate
synthase in tomato and Arabidopsis thaliana (hereafter referred
to as Arabidopsis) reduces GA content without affecting the
levels of carotenoids and chlorophylls suggests that ent-kaurene
is produced from a separate pool of GGPP, via a GGPS (GGPP
synthase) that, in contrast with the GGPS isozymes involved in
pigment synthesis, has a requirement for GPP as substrate [22].
This would require that GAs and the GGPP-derived pigments are
produced in different tissues and is consistent with ent-kaurene
synthesis in leaves occurring in immature chloroplasts associated
with the vasculature [23]. Plants contain relatively many GGPS
genes, the Arabidopsis genome containing 12 such genes, with
most of the encoded enzymes localized in plastids [24], and it
is feasible that one or more of these may be dedicated to GA
biosynthesis. The fungus F. fujikuroi contains two GGPS genes,
one of which is located in the GA biosynthesis gene cluster and
is used exclusively for GA biosynthesis [25].
Synthesis of ent-kaurene from GGPP occurs in two steps
via ent-copalyl diphosphate, the reactions catalysed by separate
enzymes, CPS (ent-copalyl diphosphate synthase), a class II
(proton-initiated) cyclase and KS (ent-kaurene synthase), a class I
(initiated by phosphate ionization) cyclase respectively, in higher
plants [26]. These reactions are also catalysed by separate CPS
and KS enzymes in the GA-producing bacterium Bradyrhizobium
japonicum [27], whereas in fungi both activities reside in a single
polypeptide [28]. In Arabidopsis, both CPS and KS are encoded
by single genes, loss of function of which results in severe GAdeficient phenotypes, characterized by extreme dwarfism, male
and female infertility and non-germinating seeds [29]. Several
species, particularly cereals, have been found to express two or
more CPS- and KS-like genes [30–33], although in rice only one
member of each gene family is thought to be involved in GA
biosynthesis, the others producing phytoalexins [30,33].
ent-Kaurene is relatively volatile and has been found
to exchange with the external environment, prompting the
suggestion that it may function as a mediator of plant–
plant communication [34]. However, since regulation of GA
biosynthesis occurs mainly at later stages of the pathway [35],
exogenous ent-kaurene is unlikely to have a major influence on
GA content and the resulting growth.
The conversion of ent-kaurene into the bioactive forms involves
the action of membrane-associated P450s (cytochrome P450
mono-oxygenases) and soluble ODDs (2-oxoglutarate-dependent
dioxygenases). The formation of GA12 , which is considered
the common precursor for all GAs in plants [36], requires six
oxidative steps, catalysed by two mono-oxygenases, KO (entkaurene oxidase) and KAO (ent-kaurenoic acid oxidase). KO
belongs to the CYP701A P450 clade and KAO to CYP88A [37].
These enzymes are localized in the endoplasmic reticulum and, in
the case of KO, also in the plastid envelope [38]. The three-step
KO-catalysed oxidation of ent-kaurene to ent-kaurenoic acid via
ent-kaurenol and ent-kaurenal has been shown to involve repeated
hydroxylations on C-19, with the initial step rate-limiting and
the intermediates retained at the enzyme active site [39]. KO is
encoded by a single gene in Arabidopsis, whereas rice contains
Regulation of gibberellin biosynthesis
Figure 2
13
The GA-biosynthetic pathway from trans -geranylgeranyl diphosphate to GA1 , GA3 and GA4
The reactions are indicated with three-dimensional structures and the functionality introduced by each oxidative step is shown in red. The subcellular compartmentalization of the pathway and the
enzymes catalysing each step are also indicated. GA13ox, GA 13-oxidase.
a cluster of five KO-like genes [30], one of which, OsKO2,
was shown by heterologous expression in yeast to possess KO
activity [40]. Mutations in OsKO2 cause severe GA-deficiency
and dwarfism, confirming its involvement in GA-biosynthesis and
suggesting it may be the only gene of the cluster to have this
role [30].
The oxidation of ent-kaurenoic acid to GA12 by KAO occurs
in three steps via the intermediates ent-7α-hydroxykaurenoic
acid and GA12 -aldehyde, requiring successive oxidations at C7β, C-6β and C-7 (evidence summarized in [41]). In fungi
and developing seeds of some plant species in which GAs
are produced at very high levels, the first two of these
reactions produce by-products (kaurenolides and fujenoic acids
respectively) in substantial quantities, but these do not appear
to be formed in tissues in which GAs are produced in hormonal
quantities. The fungus F. fujikuroi also employs cytochrome P450s
for the KO and KAO reactions, but they are unrelated to the higher
plant enzymes, belonging to the CYP503 and CYP68 clades
respectively, and appear to have evolved independently [42]. The
F. fujikuroi KAO produces mainly GA14 (3β-hydroxyGA12 ) rather
than GA12 due to 3β-hydroxylation of GA12 -aldehyde, an activity
not present in the higher plant enzymes [43].
Whereas rice contains a single KAO gene [30], Arabidopsis
contains two fully redundant copies [44]. Two KAO genes have
also been reported in pea and sunflower, but these genes differ
in expression domains, with one gene being expressed in most
tissues, whereas expression of the second gene is restricted to
developing seeds and roots [45,46]. The pea enzymes were found
to produce small amounts of the non-GA by-products in vitro [45],
perhaps in response to high substrate concentrations. Lesions in
KAO have been shown to be the cause of severe dwarfism in several
species, including rice (d35 mutant) [30], maize (dwarf3) [44], pea
(na) [45] and sunflower (dwarf2) [46]. Pumpkin contains an ODD
that oxidizes GA12 -aldehyde to GA12 [47], but orthologues of this
enzyme have not yet been found outside of the Cucurbitaceae.
In higher plants, GA12 lies at a branch-point in the pathway,
undergoing hydroxylation on C-13 and/or C-20. The nature of the
enzymatic activity responsible for 13-hydroxylation, by which
GA12 is converted into GA53 , is unclear. Both P450 and ODD
13-hydroxylase activities have been detected (reviewed in [48]),
and it has been recently reported that rice contains two P450s that
convert GA12 into GA53 [49]. Interestingly, the enzymes present
in Arabidopsis (CYP714A) and Stevia rebaudiana (CYP716D)
13-hydroxylate ent-kaurenoic acid rather than GA12 [50,51].
GA12 and GA53 are oxidized on C-20 by GA20ox (GA
20-oxidase), an ODD, converting these substrates in parallel
pathways into GA9 and GA20 respectively. This is a three- or
four-step process, depending on the mechanism for the loss of
C-20, which has not been determined [41]. In this reaction series,
the C-20 methyl group is oxidized to the alcohol and then the
c The Authors Journal compilation c 2012 Biochemical Society
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P. Hedden and S. G. Thomas
aldehyde, from which it is lost with the formation of a γ -lactone
between C-19 and C-10. The alcohol and aldehyde intermediates
accumulate in plants, the aldehydes often to relatively high levels
[52], consistent with them being released and having to rebind to
the enzyme active site for the successive oxidations. In contrast
with the accumulation of these intermediates, the intermediates in
the P450-catalysed reactions earlier in the pathway are normally
present in only trace quantities [53]. The C-20 alcohol readily
lactonizes with the C-19 carboxylic acid group, particularly
at low pH, preventing further oxidation by the GA20ox [54].
Moreover, some tissues contain a 20-oxidase activity capable
of oxidizing C-20 in its lactone form [54], although there is
little information on the nature of the enzyme responsible for
this activity. It is not known to what extent lactonization of
C-20 alcohols occurs in planta, because the acidic conditions used
during GA analysis promotes lactone formation, but the existence
of the lactone-oxidizing activity suggests that these δ-lactones
may occur naturally.
Little is known about the mechanism by which C-20 is lost,
although it appears to involve the formation of a free radical on
C-10 that reacts with the C-19 carboxylic acid to form the lactone
[55]. The finding that C-20 is lost directly as CO2 [56] necessitates
the existence of an intermediate between the C20 aldehyde and the
C19 γ -lactone product. However, no candidate intermediate has
been identified and it has been suggested that it remains bound
at the enzyme active site [41]. A side reaction from the oxidation
of the aldehyde results in the formation of a carboxylic acid.
This reaction can occur from a substantial to a negligible extent
depending on the nature of the GA20ox, and is most common
in developing seeds (see, e.g., [57]). The resulting tricarboxylic
acid product is not converted into C19 -GAs and constitutes a biologically inactive end-product. Oxidative loss of C-20 in the fungi
F. fujikuroi and Sphaceloma manihoticola is catalysed by a P450
rather than an ODD [58,59]. Apart from GA14 , the first substrate
for the fungal enzyme, the proposed C20 -GA intermediates (GA37
and GA36 ) in the formation of GA4 accumulate to a much smaller
extent in fungal cultures than do the equivalent intermediates
in plants, and, in contrast with the plant intermediates, are not
oxidized to C19 -GA products when added to fungal cultures [59].
This may indicate that these fungal intermediates do not leave
the active site, and their presence in fungal cultures may be due
to hydrolysis of enzyme-bound intermediates [41]. However,
cultures of F. fujikuroi grown in the presence of 18 O2 or H2 18 O
incorporated 18 O only from 18 O2 into the intermediates, indicating
that they were not formed by hydrolysis [60].
The final step in the formation of the biologically active
hormones is the 3β-hydroxylation of GA9 and GA20 to GA4
and GA1 respectively, catalysed by the ODD GA3ox (GA 3oxidase). In shoots of dicotyledonous species this reaction tends
to be highly regiospecific, with a single product being produced
[61–63], whereas in several monocotyledons, in addition to
GA1 , GA3 is formed from GA20 via the intermediate GA5 by
the action of a single GA3ox [64–66]. Developing seeds of
certain dicotyledonous species contain GA3oxs of relatively low
regiospecificity, possessing 2β-hydroxylase [65,67,68] and/or
2,3-desaturase [65,69,70] activities in addition to their 3βhydroxylase activity. C-2,3-unsaturated GAs may be further
oxidized by GA3oxs to the 2,3-epoxide or to the 3β-hydroxy1,2-unsaturated products. Thus GA5 may be converted into GA6
or GA3 , this latter conversion involving abstraction of the 1β-H,
rearrangement of the 2,3-double bond to the 1,2-position and
then addition of OH at C-3β [69]. Whereas both desaturation
and formation of the 3β-hydroxy-1,2-unsaturated product
are catalysed by a single enzyme in cereals, separate enzymes are
required for these reactions in developing Marah macrocarpus
c The Authors Journal compilation c 2012 Biochemical Society
seeds [70]. In an example of extreme lack of regiospecificity, two
GA3ox enzymes, from wheat [64] and M. macrocarpus seeds
[70], were shown to possess some 13-hydroxylase activity. This
requires that the substrate binds to the enzyme active site in
opposite orientations to be oxidized either on C-3 or C-13, and
indeed C19 -GA substrates, such as GA9 , possess some symmetry,
with the 6-carboxylic and 19,10-lactone moieties potentially able
to exchange positions in the binding site. Although the 13hydroxylase activity of the M. macrocarpus enzyme may have
some significance in seeds of this species, in which low amounts
of 13-hydroxy-C19 -GA, but no 13-hydroxy-C20 -GAs, are present,
consistent with this reaction occurring late in the pathway, early
13-hydroxylation by a regiospecific enzyme appears to be the
most common route in higher plants. In the fungus F. fujikuroi,
13-hydroxylation of GA7 to GA3 by a P450 is the final step in the
biosynthesis of GA3 in this species [71].
In contrast with the earlier enzymes, the ODDs are encoded
by multiple genes in all plant species that have been studied:
for example there are five GA20ox enzymes in Arabidopsis and
four in rice, whereas these species contain four and two GA3ox
genes respectively [72]. Members of these gene families vary
considerably in their levels and patterns of expression, and are
differentially regulated (see below). This multiplicity of genes
may be related to the fact that the GA biosynthetic pathway is
regulated primarily by the activity of the ODDs.
GIBBERELLIN DEACTIVATION
It is essential for plants to be able to regulate precisely their GA
content and to have the ability to change this parameter rapidly in
response to changes in their environment. In common with other
plant hormones, GAs are inactivated and this provides a means
to regulate homoeostasis and to allow a rapid reduction of GA
concentration when required. Several mechanisms for inactivating
GAs have been identified (Figure 3), the most prevalent being 2βhydroxylation. The enzymes responsible for this activity, GA2oxs
(GA 2-oxidases), are ODDs, which can be divided into two
major groups according to function: one group acts on C19 -GAs,
including the bioactive compounds and their immediate non-3βhydroxylated precursors [73], whereas another group acts on C20 GAs [74]. The C19 -GA2oxs can be divided into two subgroups on
the basis of amino acid sequence [75–77] and it has been proposed
that they differ also in biochemical function [76]. Several C19 GA2oxs, all of which belong to the same subgroup, have been
shown to be bifunctional, acting as 2β-hydroxylases, but also
further oxidizing the 2β-hydroxy group to a ketone. The isolated
2-keto products, known as GA-catabolites [78], are dicarboxylic
acids with a double bond at C-1,10, C-5,10 or C-10,11 (shown for
the 1,10 isomer in Figure 3) that are thought to arise from the 2ketone 19,10-lactones by rearrangement, probably during analysis
[42]. The catabolites can accumulate to high concentrations
in seeds of some species, for example, pea [79]. The second
subgroup of C19 -GA2oxs do not appear to possess the capacity
to produce the GA-catabolites [76]. The GA2ox enzymes form
particularly large gene families, although, on the basis of sequence
phylogeny, the C20 -GA2oxs might be considered a separate family
from those acting on C19 -GAs [80]. In common with the other
ODDs, the genes encoding GA2oxs are major sites of regulation
(see below).
Other mechanisms for GA deactivation have been recognized in
recent years. A cytochrome P450 mono-oxygenase (CYP714D1),
which is encoded by the EUI (ELONGATED UPPERMOST
INTERNODE) gene in rice, was shown in vitro to convert GAs
into their 16α,17-epoxides [81]. The enzyme accepted GA12 ,
Regulation of gibberellin biosynthesis
Figure 3
15
Reactions that result in GA deactivation
Purple arrows denote reactions catalysed by C20 -GA2oxs, whereas reactions represented by red arrows are catalysed by C19 -GA2oxs. Formation of the 16α, 17-dihydrodiols is initiated by the
epoxidase EUI (green arrows). Gibberellin methyl esters are formed in developing seeds of Arabidopsis by SAMT methyl transferases (blue arrows). The brown arrows indicate reversible formation
of GA glucosyl esters. The functionality introduced in each case is indicated by the appropriate colour.
GA9 and GA4 as substrates (other 13-desoxyGAs were not
tested), but was much less effective with 13-hydroxylated GAs.
Overexpression of EUI in rice caused extreme dwarfism and a
reduction in GA4 content in the uppermost internode, confirming
that epoxidation resulted in GA deactivation. However, it was
not possible to identify the epoxides in plant tissues, even
when EUI was overexpressed, but the accumulation of GA16α,17-dihydrodiols indicates that the epoxides are hydrated
in planta, either enzymatically or by a non-enzymatic acidcatalysed reaction. GA dihydrodiols have been found to occur
naturally in many plant species (discussed in [81]), suggesting
that epoxidation may be a general deactivation mechanism.
Arabidopsis contains two CYP714 enzymes, one of which,
CYP714A2, 13-hydroxylates ent-kaurenoic acid to steviol [50].
Overexpression of both CYP714 genes resulted in dwarfism, with
CYP714A1 producing the most severe effect [82], although the
function of its encoded enzyme has not been reported.
Incorporation of an eui mutant allele into MS (male-sterile)
rice varieties is used to improve their heading performance [83].
MS varieties used in hybrid rice breeding suffered from failure
of the panicle to emerge fully from the leaf sheaths, a syndrome
that was effectively overcome by the extended upper internodes
resulting from the presence of the eui mutation. The effect of this
mutation is consistent with the expression pattern of EUI, which,
on the basis of reporter gene analysis, occurs most strongly in the
flowering spikelets and in the nodes and cell division zone of
the expanding internodes [81]. Rice anthers produce extremely
high levels of GA4 [84], and it is possible that EUI may regulate
the influx of GA4 into the stem from reproductive tissues, whereas
GA1 produced in vegetative tissues [85] would be unaffected.
Two members of the SABATH group of methyl transferases,
named GAMT (GA methyl transferase) 1 and GAMT2, present
in Arabidopsis, were shown to be specific for GAs [86,87]. The
enzymes methylate the 6-carboxy group of C19 -GAs, a process that
abolishes biological activity [88]. Thus ectopic overexpression of
these genes in Arabidopsis resulted in dwarfism. The GAMT genes
are expressed in developing seeds, where they apparently function
to regulate GA content, since gene knockout lines accumulated
GAs [86]. Their physiological roles may be restricted to seed
development and the control of precocious germination.
GIBBERELLIN CONJUGATES
Ester and ether GA conjugates have been identified in a range of
plant species, with glucose the predominant conjugate partner
[89]. Although there has been interest in these compounds
as potential storage and/or transport forms of GA, it was
difficult to assign to them a definite physiological role, and
attention to this topic has waned in recent years. Consequently,
there is limited information on the enzymes involved in their
formation or hydrolysis. Some GA conjugates were shown
to have biological activity in bioassays, but this was thought to
be due to hydrolysis to the aglycone in the assay system [90].
Evidence for the reversibility of GA conjugate formation adds
to the feasibility of their having a storage role [91], providing
a mechanism for rapid release of bioactive GA that would,
for example, stimulate seed germination. A recent report that
ectopic expression of a β-glucosidase in transplastomic tobacco
resulted in increased levels of several hormones including GA1
and GA4 [92] suggests that plastids may act as a store of
hormone conjugates. Conjugation might also provide a potentially
reversible deactivation mechanism, although evidence for this
is lacking. It is of interest that 2β-hydroxyGAs form glucosyl
ethers linked through the 2β-hydroxyl group [89]. In this case,
conjugation may serve to solubilize and trap these inactive endproducts in the vacuole.
REGULATION OF GA BIOSYNTHESIS
Sites of synthesis
In general, the highest levels of bioactive GAs are found in actively
growing organs, for example developing leaves and expanding
internodes [93]. Such organs contain high expression levels of
c The Authors Journal compilation c 2012 Biochemical Society
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P. Hedden and S. G. Thomas
GA-biosynthetic genes, indicating active GA synthesis [23,94].
On the basis of the coincidence of expression of GA20ox and
GA3ox genes with that of the GA signal transduction gene SLR1
(SLENDER1) in growing organs of rice, Kaneko et al. [94]
concluded that GAs are synthesized at their site of action in such
organs. There are several examples of different reactions of the
pathway occurring in separate tissues, requiring the movement
of intermediates between cells. On the basis of promoter–GUS
(β-glucuronidase) reporter analysis and in situ hybridization,
Yamaguchi et al. [95] found that, whereas CPS was expressed
in the provasculature of the embryonic axis in germinating
Arabidopsis seeds, KO and GA3ox genes were expressed in the
cortex and endodermis. This would require the movement of an
intermediate, potentially ent-kaurene, from the provasculature, a
highly plausible scenario given the demonstrated release of this
intermediate into the environment [34]. The synthesis of entkaurene from GGPP can be considered the gateway into the GA
biosynthetic pathway and as such would control the metabolic
flux into the pathway [23]. On the basis of incubations with cellfree systems from wheat tissues, Aach et al. [20] concluded that
ent-kaurene is synthesized in proplastids within regions of cell
division, but not in fully developed chloroplasts in elongating or
mature cells. In the stem, they found the highest activity in the
node, assumed to be associated with the intercalary meristem.
This region was also reported to be the major site of TaGA20ox1
expression in the upper wheat stem, with TaGA3ox1 expression
divided more evenly between the nodes and internodes [64,96].
Since cell elongation in the internode requires GA signalling
[97], this distribution of activities would require movement of
GAs or precursors from the meristem to the elongation zone, or
alternatively GAs and intermediates may remain in the dividing
cells as they move into the elongation zone. In contrast with this
result, Kaneko et al. [94] reported promoter–GUS expression for
the rice GA20ox2 paralogue in both the node and elongation zone,
indicating marked differences between these monocot species.
In addition to growing tissues, CPS–GUS staining was found by
Silverstone et al. [98] in the vasculature of mature leaves, in which
the chloroplasts are immature and may thus be still capable of
ent-kaurene synthesis. Indeed, Ross et al. [99] demonstrated that
mature pea leaves and internodes were active for GA biosynthesis,
but contained low levels of GA1 and GA20 due to high rates of
2β-hydroxylation. The potential association of GA biosynthesis
with the vasculature raises the possibility that this tissue may
act as a source of GAs for export via the phloem. There is in
fact considerable evidence from experiments involving grafting
and application of labelled GAs for long-distance movement of
GAs in the vasculature (reviewed in [100]). Results from such
experiments with pea [101] indicated that GA20 rather than GA1
or earlier intermediates is the mobile form in this species. It has
been demonstrated in a number of species that leaves produce
GAs as mobile signals for induction of flowering. In Arabidopsis,
flowering in short days was associated with an increase in GA4
concentration at the shoot apex, assumed to have been exported
from the leaves [102], whereas in Lolium species, the mobile
signal, produced in the leaves in response to long days, was
suggested to be GA5 and GA6 , which would escape metabolism by
GA2oxs [103]. A discussion of the regulation of GA biosynthesis
by photoperiod is given below.
Certain tissues, which are particularly active in GA
biosynthesis, function as sources of GA to support the
development of neighbouring tissues. In germinating cereal
grains, the scutellum epithelium exports GAs to the non-GAproducing aleurone cells [94], which respond by synthesizing
and secreting hydrolases to the endosperm as well as undergoing
PCD (programmed cell death) [104]. Within flowers, the highest
c The Authors Journal compilation c 2012 Biochemical Society
concentrations of GAs occur in anthers, in which the tapetum
has been identified as an important site of synthesis [84,105].
Interestingly, the tapetum also undergoes PCD in response to
GA, to release components that are incorporated into the pollen
wall [106]. Some flower organs that are non-autonomous for
GAs, particularly the petals, are dependent on the anthers,
and potentially also the receptacle, to supply these hormones
for normal development [105,107]. The suspensor, an organ
present in the seed during early embryogenesis that supports
growth of the young embryo [108], has been shown in several
species to be an extremely rich source of GAs, which are
thought to be important for early embryo growth (reviewed
in [109]). Later in development, the immature seed of many,
but not all, species contain high levels of GAs, which may
be produced in the endosperm and/or the cotyledons. These
organs can produce a vast range of different GA structures
not found in other tissues [110], the function of which is
unclear. A characteristic of these high-GA-producing tissues is the
recruitment of several ODD paralogues. For example, in the rice
scutellum and tapetum, OsGA20ox1 and OsGA3ox1 are expressed
in addition to OsGA20ox2 and OsGA3ox2, the first two genes
not being expressed elsewhere [94]. Tissues of developing seeds
typically express genes not active in other tissues, as for example
found in pumpkin [111]. These genes are presumably not subject
to the regulatory constraints imposed by the normal mechanism
for GA homoeostasis (see below).
Developmental regulation
Consistent with its proposed role as the ‘gatekeeper’ for GA
biosynthesis, CPS expression is highly developmentally regulated
as noted above [23]. Although CPS activity may regulate
the flux through the GA biosynthetic pathway, the activity
of the ODDs more precisely determines the concentration of
bioactive GA [35]. The different ODD paralogous genes typically
show distinct, but overlapping, expression patterns and as such
contribute unequally to different developmental processes. For
example, in Arabidopsis, loss of AtGA20ox1, but of no other
individual GA20ox, results in a reduction in stem internode length,
whereas additional loss of AtGA20ox2 causes further internode
shortening, indicating that the latter also contributes to internode length [112]. However, this contribution is exaggerated in
the absence of AtGA20ox1, due to compensatory mechanisms
(see below). A similar relationship exists between AtGA3ox1
and AtGA3ox2 in the regulation of stem extension [113].
Some indications of the molecular mechanisms underlying
the developmental regulation of GA biosynthesis have been
reported. The LEC (LEAFY COTYLEDON) transcription factors
LEC2 and FUS3 (FUSCA3), which are required for correct
tissue specification during embryogenesis and for normal seed
development, suppress GA biosynthesis in the developing embryo
by down-regulating AtGA3ox2 expression in the epidermis [114].
FUS3 was shown to bind to RY elements in the AtGA3ox2
promoter. The requirement to restrict GA production during
embryogenesis is reinforced by the findings of Wang et al.
[115], who reported that the GA-deactivation gene AtGA2ox6 was
up-regulated in the developing embryo by the MADS [MCM1
(minichromosome maintenance 1), AGAMOUS, DEFICIENS
and SRF (serum-response factor)] domain transcription factor
AGAMOUS-LIKE 15. Unlike most other organs of Arabidopsis,
developing seeds contain higher amounts of GA1 than GA4 ,
indicating that 13-hydroxylase activity is high specifically at this
stage of development [114]. Another MADS domain protein
AG (AGAMOUS) was shown to directly promote expression
of AtGA3ox1 in developing flowers [116]. AG function is
Regulation of gibberellin biosynthesis
necessary for correct floral organ specification, but also for their
development, and although organ specification can occur in the
absence of GA signalling [117], GAs are necessary for floral organ
development [118]. AtGA3ox1 was also shown to be a direct target
of the bHLH transcription factor INDETERMINATE in the valve
margins and septum of the Arabidopsis silique, as part of the
process of pod opening and seed dispersal [119].
In the vegetative shoot apex, GA biosynthesis occurs in leaf
primordia and in the elongating sub-apical region [120]. However,
in order to maintain a pool of indeterminant cells in the shoot
apical meristem, it is proposed that bioactive GAs must be
excluded from this region through the action of homeodomain
transcription factors of the Class-I KNOX (KNOTTED-like
homeobox) class, which suppress the expression of GA20ox genes
[120,121]. The tobacco homeobox NTH15 (Nicotiana tabacum
homeobox 15) was shown to interact with a sequence within the
first intron of the NtGA20ox1 gene [121]. In potato, suppression of
StGA20ox1 expression was mediated by both the KNOX protein
POTH1 and a BEL-type homeodomain protein, StBEL5, which
bound as a heterodimer to the promoter [122]. The expression of
GA2ox genes has been localized to the base of the meristem
in several species where the enzyme may function to protect
the meristem from the influx of GAs [123–126]. Expression
of the rice gene OsGA2ox1 is reduced when plants are grown
in florally inductive short days, so permitting an influx of GAs
for promotion of the floral transition [125]. In Arabidopsis,
expression of AtGA2ox2 and AtGA2ox4 was up-regulated by
induction of the KNOX protein SHOOTMERISTEMLESS and
by cytokinin, which was proposed to mediate this effect through
KNOX-induced expression of the cytokinin-biosynthetic gene
ISOPENTENYLTRANSFERASE [124]. However, direct induction
of GA2ox expression by KNOX was demonstrated in maize, with
the homeodomain protein KNOTTED1 binding to the first intron
of ZmGA2ox1 [123].
Regulation of GA biosynthesis by other hormones
There has been considerable interest in recent years in interactions
between hormone signalling pathways [127]. Such interactions
are consistent with the overlapping physiological roles found for
the different hormones, although this is not so clearly reflected
at the molecular level, with, in some cases, the hormones
regulating different, albeit functionally related, genes [128].
Interactions through regulation of hormone biosynthesis have
been invoked in a number of cases, although direct evidence for
this is rarely presented and its occurrence may be exaggerated
[129]. However, there is strong evidence for regulation of GA
concentration by auxin signalling at the level of ODD gene
expression, with up-regulation of GA20ox and/or GA3ox genes
and, in some cases, down-regulation of GA2ox genes resulting
in increased GA content [130–134]. The molecular mechanism
for auxin regulation of GA-biosynthesis gene expression is not
known, but appears to be direct and does not involve DELLA
proteins [130,132], although the effect may be masked by
DELLA-mediated homoeostasis in some cases [135].
Although Ross et al. [129] could find no evidence for regulation
of GA metabolism by ABA (abscisic acid) in pea internodes, an
interaction between these hormones has been shown in the context
of seed germination [136]. The CHO1 (CHOTTO1) transcription
factor, which enhances dormancy in imbibed Arabidopsis seeds,
inhibits GA biosynthesis through suppression of AtGA3ox2
expression [136]. Since expression of CHO1 requires ABA
signalling [137], this transcription factor couples the regulation of
GA biosynthesis to ABA signalling. There has been no evidence
17
that CHO1 binds directly to the AtGA3ox2 promoter, so the
regulation may be indirect or involve interaction with other
transcription factors, either scenario being consistent with this
regulation being specific to imbibed seeds despite expression of
CHO1 occurring in other organs [136].
Gibberellin homoeostasis
Even prior to the identification of genes encoding GA metabolic
enzymes or GA signalling components, it was apparent that
homoeostatic mechanism(s) existed to maintain optimal GA levels
for co-ordinated plant growth and development. The present
review will focus on recent advances in understanding the
molecular events involved in this mechanism, summarized in
Figure 4. However, it is important to acknowledge that it is
part of a wider homoeostatic mechanism, also including the
regulation of transcription of core GA signalling components
[13,14,112,117,138,139], which potentially co-ordinates GAresponsive growth through maintenance of optimal levels of the
growth-repressing DELLA proteins.
The biochemical study of GA biosynthetic and response
mutants in several plant species demonstrated that GA levels are
controlled through a process of feedback regulation (reviewed
in [140]). The subsequent cloning of genes encoding GA
biosynthesis enzymes led to the demonstration that this regulation
was mediated through the transcriptional control of specific
ODDs. For example, in Arabidopsis this includes three GA20ox
genes (GA20ox1, GA20ox2 and GA20ox3) and a single GA3ox
(GA3ox1) whose expression levels are all highly elevated when
bioactive GA levels are low, but are then rapidly reduced when
these plants are treated with exogenous GA [112,113,141,142].
In contrast, other members of these two gene families, in addition
to those encoding enzymes catalysing all of the earlier steps in the
GA biosynthetic pathway, do not appear to be subject to feedback
regulation [112,113,143]. An additional level of regulation that
functions in GA homoeostasis involves the feedforward regulation
of GA2ox genes [73,75,138]. Studies in Arabidopsis have
demonstrated that multiple members of both the C19 and C20
classes of GA2oxs are transcriptionally up-regulated in response
to increased GA signalling output [73,138,144]. On the basis of
these observations it is likely that GA-mediated transcriptional
changes in the ODD genes translate to alterations in the levels
of the metabolic enzymes they encode, ultimately leading to the
changes in flux of GAs through this pathway that are necessary
for controlling the levels of bioactive GAs. The low abundance
and labile nature of these metabolic enzymes has hindered their
detection and the subsequent confirmation of this hypothesis. An
exception to this was a recent study of the photoperiodic regulation
of a GA20ox gene in spinach, in which the authors were able to
detect the endogenous SoGA20ox1 protein by Western blotting
[145]. Although they found no evidence for the transcriptional
feedback regulation of this gene in petioles and shoot tips of
plants treated with GA biosynthesis inhibitors, these treatments
resulted in a clear increase in SoGA20ox1 protein levels. These
findings allude to further complexities in GA homoeostasis
through potential post-transcriptional regulatory mechanisms.
In light of the importance of targeted protein degradation in
regulating GA signal transduction, it is tempting to speculate
that similar mechanisms may exist to control the regulation of
GA metabolism. A precedence for this is the feedback regulation
of ACC synthase stability by ethylene signalling [146].
Recent advances in our understanding of GA signal
transduction have provided important insights into the
mechanisms controlling GA homoeostasis. There is conclusive
evidence demonstrating that transcriptional feedback and
c The Authors Journal compilation c 2012 Biochemical Society
18
Figure 4
P. Hedden and S. G. Thomas
Homoeostatic regulation of GA metabolism and signalling
In the absence of GA signalling, DELLAs increase GA levels through transcriptional control of GA
metabolic genes. The subsequent increase in GA concentration leads to GID1-mediated DELLA
degradation, allowing GA homoeostasis. The homoeostatic control mechanism also includes
the transcriptional regulation of GA signalling genes by DELLAs. SCL3 acts to attenuate DELLA
activity through direct association. The shaded box indicates transcription factors that have a
potential role in controlling the transcriptional feedback regulation of specific GA biosynthesis
genes. Their relationship to the regulation of these genes by DELLAs is currently unclear. Grey
lines indicate transcriptional control.
feedforward regulation of GA metabolism is mediated by the
GA signalling pathway and is dependent on the levels of
DELLA proteins. In the case of feedback regulation, this has
been clearly illustrated in mutants lacking core GA signalling
components including GID1 [8,117,147,148] and GID2/SLY1
(SLEEPY1) [149,150], which are necessary for targeting DELLA
degradation in response to GA. In these mutants, the expression
levels of feedback-regulated GA biosynthetic genes are highly
elevated and are not down-regulated by exogenous GA treatment.
Consequently, these mutants accumulate highly elevated levels
of bioactive GAs [8,117,147,150]. The absolute requirement
for DELLAs in controlling the feedback response has also
been demonstrated extensively in many higher plant species
by characterizing both loss-of-function and gain-of-function (in
which the abnormal DELLAs are resistant to GA-mediated
degradation) mutants. Notably, it was the study of DELLA
gain-of-function mutants in maize, wheat and Arabidopsis that
initially provided the evidence for feedback regulation of GA
biosynthesis on the basis of the observations that they contained
elevated levels of bioactive GAs [151–153]. Subsequently, it has
been demonstrated that the transcriptional feedback regulation is
perturbed in both classes of DELLA mutants, with transcript levels
found to be low in those lacking DELLAs, but highly elevated in
those containing stabilized forms [144,154–157]. Most of these
studies have focused on the role of DELLAs in the feedback
regulation of GA20ox and GA3ox. However, a recent study of a
DELLA loss-of-function mutant in pea (la cry) has demonstrated
that the GA-mediated feedforward regulation of PsGA2ox1 and
PsGA2ox2 is also controlled by these DELLAs [144].
DELLAs are nuclear-localized proteins that function as
transcriptional regulators [9,156,158,159]. Because they act
as growth repressors, there is occasionally the misconceived
perception that they are transcriptional repressors. In fact, an
early study of the rice DELLA SLR1 suggested that it acts
as a transcriptional activator in yeast [159a]. This was later
confirmed in a detailed transcriptomics analysis of DELLA
target genes in Arabidopsis, showing that the majority of
c The Authors Journal compilation c 2012 Biochemical Society
these are up-regulated by DELLA (and down-regulated by GA)
[9]. Interestingly, those genes showing the largest changes in
expression in response to alterations in DELLA levels included
the feedback-regulated genes GA20ox2 and GA3ox1. This work
and an earlier study by Gubler et al. [160] demonstrated that
DELLA protein degradation occurs within 5–10 min following
GA treatment of GA-deficient Arabidopsis seedlings and barley
aleurone layers [9,160]. In Arabidopsis, the subsequent reductions
in the transcript levels of GA3ox1 and GA20ox2 were detectable
after 15 min, suggesting that these are probably DELLA primary
target genes [9]. However, Zentella et al. [9] were unable to detect
binding of an RGA (REPRESSOR OF ga1)–TAP (tandem affinity
purification)-tag fusion protein to the promoters of GA3ox1 or
GA20ox2, suggesting that other factors are necessary to regulate
transcription.
There has been considerable interest in attempting to
understand the mechanisms and identify the transcription factors
that are responsible for controlling the feedback regulation of GA
biosynthesis. Despite these efforts the picture is still rather unclear,
although several transcription factors that appear to have a role
in this process have been identified (recently reviewed in [161]).
The most well characterized of these transcription factors is RSG
(REPRESSION OF SHOOT GROWTH), a bZIP (basic leucine
zipper)-containing transcription factor identified in tobacco [162].
The characterization of a dominant-negative form of RSG initially
demonstrated that it acts as a transcriptional activator of NtKO.
It was subsequently demonstrated that RSG also binds to and
activates the expression of the feedback-regulated NtGA20ox1
gene [163]. Binding of RSG to the NtGA20ox1 promoter was
found to be rapidly suppressed by GA, indicating that it might
control the transcriptional feedback regulation of this gene.
Interestingly, there was no evidence that RSG controlled the GAmediated feedback regulation of another GA biosynthetic gene,
NtGA3ox [164].
The identification of RSG protein interactors has led to
some important findings about the regulatory mechanisms
controlling RSG transcriptional activity. These comprehensive
studies have demonstrated that 14-3-3 proteins bind to RSG,
sequestering it in a cytoplasmic localization in response to GA
signalling and preventing its activation of NtGA20ox1 and other
target genes [164,165]. This process is controlled by CDPK1
(Ca2 + -dependent protein kinase 1), which in response to GA
signalling, and potentially an elevation in intracellular Ca2 +
levels, phosphorylates RSG on Ser114 , enhancing its association
with 14-3-3 proteins in the cytoplasm [164,166]. Although
these studies provided important insights into the control of
feedback regulation of NtGA20ox1, there are still many remaining
questions. Notably, the role of DELLAs in the regulation of
RSG activity is unclear. It is conceivable that DELLA signalling
regulates intracellular Ca2 + levels, and indeed an increase in Ca2 +
levels is associated with GA signalling in the cereal aleurone
[167]. A similar function for RSG orthologues in the regulation
of GA biosynthesis has not been reported for other plant species,
but if this turns out to be the case, the availability of improved
genetic resources in model species should provide important tools
for dissecting the mechanisms involved.
In Arabidopsis, characterization of the cis-acting sequences
responsible for controlling the feedback regulation of AtGA3ox1
has led to the identification of AGF1 (AT-hook protein of GA
feedback 1) as a potential regulator of this response [168]. The
presence of an AT-hook DNA-binding motif together with the
demonstration that an AGF1–GFP (green fluorescent protein)
fusion protein is nuclear-localized in transgenic plants supports a
role of AGF1 as a transcriptional regulator. However, although the
overexpression of AGF1 potentiates the transcriptional feedback
Regulation of gibberellin biosynthesis
regulation of AtGA3ox1, a clear role in regulating this response
has yet to be established.
Members of the YABBY family of C2 C2 zinc finger
transcription factors have a well characterized role in the
maintenance of polar outgrowth of lateral organs [169]. Although
extensive studies of the YABBY genes in Arabidopsis have not
yet indicated involvement in the regulation of GA metabolism,
there is evidence in rice implicating OsYAB1 (OsYABBY1) in
the feedback control of OsGA3ox2 [170]. OsYAB1 was shown to
directly repress the expression of this gene, potentially by binding
to a GA-responsive cis-regulatory element within its promoter.
The expression of OsYAB1 transcripts was found to increase
rapidly in rice seedlings after GA treatment, with paclobutrazol
treatment having the opposite effect. These observations suggest
that the GA signalling-mediated changes in OsYAB1 expression
are responsible for regulating OsGA3ox2 transcript levels as
opposed to a direct effect of OsYAB1 on DELLA (SLR1)mediated transcription. Interestingly, the expression of another
GA-regulated gene, OsGA20ox2, did not appear to be controlled
by OsYAB1, supporting the existence of additional components
responsible for its feedback regulation.
Important recent studies have identified SCL3 (SCARECROWLIKE 3) as a novel GA signalling component that also has
a function in regulating feedback control of GA biosynthesis
in Arabidopsis [171,172]. Although SCL3 was identified as an
early GA-response gene that is positively regulated by DELLAs
through direct binding to its promoter [9], genetic analysis
demonstrates that it acts as a positive regulator of GA-responsive
root and shoot growth [171,172]. Consistent with this role,
transcript levels of the GA feedback-regulated genes GA20ox1,
GA20ox2, GA20ox3 and GA3ox1 are all elevated in the scl3
mutant [171]. Similarly, the truncated SCL3 transcript produced
in this T-DNA (transferred DNA) insertion mutant (upstream of
the T-DNA insertion) is also found to be elevated compared with
wild-type plants. These observations, and the important finding
that DELLAs physically interact with SCL3, have led to a model
in which SCL3 is proposed to attenuate the transcriptional activity
of DELLAs through direct interaction. Like DELLAs, SCL3
is a member of the GRAS family of transcriptional regulators,
but its mode of action is currently unclear. It is interesting
that SCL3 regulation of GA-dependent root growth is mediated
through its confined localization within the endodermis [172].
This is consistent with studies demonstrating that GA signalling
within the endodermis drives the cell elongation and cell division
necessary for promoting GA-dependent root growth [173,174].
However, it is not entirely consistent with a role in controlling
the feedback regulation of GA biosynthesis because some of
these genes, including GA20ox1, GA20ox2 and GA3ox1, are
predominantly expressed in other cell types within the root
[175,175a]. Further work is necessary to understand the
mechanisms involved.
How an SCL3/DELLA complex acts to regulate the expression
of direct target genes is still unclear. ChIP (chromatin
immunoprecipitation) studies suggest that this complex binds
directly to regulate the expression of the SCL3 promoter [9,171],
although this does not appear to be the case for the GA feedbackregulated genes GA20ox1, GA20ox2, GA20ox3 and GA3ox1. It
is conceivable that regulation of these genes occurs through an
alternative mechanism, for example, through the sequestration
of a transcriptional repressor. The characterization of DELLAinteracting proteins is rapidly improving our understanding of
how they control growth regulation, and it is likely that the
identification of others will further improve this knowledge and
potentially answer the question of how they control the expression
of genes controlling GA homoeostasis.
19
Environmental regulation
In common with other phytohormones, GAs act as mediators of
environmental signals, allowing plants to respond, often rapidly,
to changes in light conditions, temperature, water and nutrient
status, and other abiotic and biotic stresses. In many cases it has
been shown that changes in the environment result in altered
GA content and the molecular mechanisms underlying these
metabolic changes are beginning to be resolved, primarily in
relation to light and abiotic stress.
Depending on context, GA metabolism is sensitive to changes in
light quantity, quality or duration, which may result in increased
or decreased GA content. In early seedling development, GA
signalling suppresses photomorphogenesis [176], with exposure
of dark-grown seedlings to light, i.e. de-etiolation, resulting
in a rapid reduction in GA content [177]. Both phytochrome
and cryptochrome light receptors are involved in this process,
which results in up-regulation of GA2ox expression and, in
the case of blue light, down-regulation of GA20ox and GA3ox
genes (reviewed in [178]). Experiments with pea indicate
that the rapid decrease in GA1 content in etiolated seedlings
following exposure to light, due mainly to up-regulation
of PsGA2ox2 expression, required involvement of the bZIP
transcription factor LONG1, which is related to Arabidopsis HY5
(ELONGATED HYPOCOTYL 5), and, like HY5, appears to be
regulated at the level of protein stability by the COP1 (CONSTITUTIVELY PHOTOMORPHOGENIC 1) orthologue LIP1
(LIGHT-INDEPENDENT PHOTOMORPHOGENESIS 1) [179].
Gibberellins promote flowering in LD (long day) species,
in which exposure to LDs enhances GA production, primarily
through up-regulation of GA20ox genes (reviewed in [180]).
In Arabidopsis, up-regulation of AtGA20ox2 expression in
the leaf petiole following exposure to far-red-rich LDs, in a
phytochrome B-mediated process, is associated with enhanced
petiole elongation [181] and floral induction [182]. Although
light-induced flowering in Arabidopsis on LDs involves mainly
the CONSTANS pathway, which is independent of GAs, in the
absence of this pathway on SDs (short days), floral induction
is absolutely dependent on GAs [183]. There is evidence from
Arabidopsis to suggest that, under SDs, GA4 may function as a
mobile signal from leaves to the shoot apex for floral induction
[102], whereas in the grass Lolium temulentum, it is proposed
that the mobile signals are GA5 and GA6 , produced in leaves
in response to as little as one LD [184]. On account of their
unsaturation on C-2, these last GAs escape deactivation by
GA2oxs present below the shoot apex; following floral induction,
GA2ox activity subsides and the C-2-saturated GAs, GA1
and GA4 , are produced for promotion of floral development and
elongation of the stem [126]. Compared with GA5 , applied GA1
and GA4 are only weakly florigenic in L. temulentum, presumably
due to their rapid deactivation at the shoot apex [126]. In rice, a
SD flowering plant, following exposure to SDs, GA2ox expression
below the meristem is reduced, suggested to allow influx of GA1
from leaves to promote floral induction [125]. In an analogous,
but converse, process, SD induction of tuberization in potatoes,
a process that is inhibited by GAs, is associated with enhanced
expression of StGA2ox1 adjacent to the stolon meristem prior to
stolon swelling and tuber formation [185]. Transfer of potatoes
to SDs also results in reduced expression of StGA3ox2 in stolons,
but increased expression of this gene in shoots [186]. It is suggested that in SDs less GA is transported from the shoots to the stolons due to enhanced conversion of the mobile GA20 into the less
mobile GA1 , which accumulates and promotes shoot growth.
The red-light-induced germination of photoblastic seeds
involves accumulation of GA through enhanced synthesis
c The Authors Journal compilation c 2012 Biochemical Society
20
P. Hedden and S. G. Thomas
Regulation of seed germination in Arabidopsis by red light and
Figure 6 Inhibition of growth in Arabidopsis by salt and cold stress through
induction of GA deactivation
The bHLH transcription factor PIL5, acting via the transcription factors DAG1 and SOM,
suppresses germination by inhibiting GA biosynthesis and promoting GA deactivation, thereby
stabilizing DELLA proteins. Red light induces PIL5 degradation via phytochrome-catalysed
phosphorylation. Stratification (cold treatment) acts to promote GA biosynthesis by preventing
suppression by the bHLH transcription factor SPT. The dark grey lines indicate feedback
regulation of GA biosynthesis and deactivation by DELLA.
Salt and cold, acting via the DREB/CBF-type transcription factors DDF1 and CBF1, respectively,
as well as others, promote expression of GA2ox genes, resulting in attenuated GA concentration
and greater DELLA stability. The dark grey arrows denote transcriptional promotion of the DELLA
gene RGL3 by DREB/CBF-type transcription factors.
Figure 5
cold
and suppressed deactivation as well as a reduction in ABA
content [187]. In Arabidopsis, this process is mediated by the
bHLH transcription factor PIL5 (PIF3-LIKE 5), an inhibitor
of germination that is degraded by the 26S proteasome after
being targeted by phosphorylation through interaction with
phytochromes A and B [188]. PIL5 suppresses GA accumulation
(and ABA degradation) by inhibiting expression of the GA
biosynthetic genes AtGA3ox1 and AtGA3ox2, while promoting
expression of the GA-deactivating AtGA2ox2, its action being
relieved by the light-stimulated degradation (Figure 5). Two
intermediaries in this pathway have been identified: the action
of SOM (SOMNUS), a CCCH-type zinc finger protein, has the
same effect on the GA metabolic genes as PIL5, which directly
induces its expression [189], whereas DAG1 (DOF AFFECTING
GERMINATION 1) inhibits expression of AtGA3ox1, but has no
effect on expression of AtGA3ox2 and AtGA2ox2 [190]. DAG1,
expression of which is induced indirectly by PIL5, was shown to
bind to the AtGA3ox1 promoter. AtGA3ox1 is also the target of
cold induction of germination (stratification) [191], expression
of the gene being suppressed in non-stratified seeds in the
dark through the action of the light-stable bHLH protein SPT
(SPATULA), although the mechanism is unclear [192].
Among the complex responses of plants to stress, growth
reduction is a common strategy that allows resources to be
focused on withstanding the stress [193]. Many hormone
signalling pathways have been implicated in stress responses,
including the accumulation of DELLA proteins, which restrain
growth and promote stress resistance [194], although it is
unclear to what extent these responses are causally related.
An emerging theme is stress-induced up-regulation of GA2ox
gene expression as a means of reducing GA content and
allowing DELLA accumulation. Response to abiotic stress is
mediated by the CBF (C-repeat-binding factor)/DREB [DRE
(dehydration-response element)-binding] transcription factors,
overexpression of which confers a degree of stress tolerance,
but also causes severe growth restriction [195]. In Arabidopsis,
c The Authors Journal compilation c 2012 Biochemical Society
high salt-induced expression of six GA2ox genes, one of which,
AtGA2ox7, was up-regulated specifically by the salt-responsive
CBF/DREB1 protein DDF1 (DWARF AND DELAYED
FLOWERING 1) [196]. DDF1 was shown to bind to a DRElike motif on the promoter of AtGA2ox7. Short-term exposure
of Arabidopsis plants to cold resulted in increased expression of
three GA2ox genes, two of which, AtGA2ox3 and AtGA2ox6,
were also up-regulated by overexpression of the cold-responsive
CBF1/DREB1b gene [197]. Cold and CBF1 overexpression also
increased expression of several GA biosynthetic genes, but this
effect was abolished in a DELLA-deficient mutant, indicating that
it occurred indirectly due to feedback regulation. Although both
salt and cold stress stimulated expression of many GA2ox genes,
the tested CBF/DREB transcription factors mediated induction
of only subsets of these, indicating a high level of specificity and
presumably the involvement of other transcription factors. As well
as the accumulation of DELLA proteins resulting from reduced
GA concentrations, it was shown that both salt and cold treatments
up-regulated expression of RGL3 (RGA-LIKE PROTEIN 3),
which encodes one of the five Arabidopsis DELLA proteins, the
induction being mediated by the respective CBF/DREB factors
[196,197]. Thus abiotic stress allows DELLAs to accumulate
through protein stabilization and enhanced gene expression.
Seed dormancy in Arabidopsis in response to low temperature
was shown recently to be associated with increased expression of
AtGA2ox6, a process which is mediated by the dormancy factor
DOG1 (DELAY OF GERMINATION 1) [198]. Interestingly,
both DOG1 and AtGA2ox6, as well as other GA2ox genes, were
regulated by CBF1 in developing seeds, but this transcription
factor was not cold-induced in this context, although it appeared
to have a role in seed dormancy.
The positive growth response of plants to increasing ambient
temperature was shown to be mediated by several hormone
pathways, including GA signalling, with AtGA3ox1, AtGA20ox1
and AtGA2ox1 identified as targets in Arabidopsis seedlings
[199]. Plant growth is reduced under abnormal thermoperiodic
conditions, i.e. lower temperatures during the light period than
Regulation of gibberellin biosynthesis
during the dark, a condition known as negative DIF (difference
between day temperature and night temperature) that is exploited
for control of plant stature [200]. Stavang et al. [201] showed
that stems of pea plants grown in negative DIF contained lower
concentrations of C19 -GAs and increased expression of PsGA2ox2
compared with plants grown in positive DIF and suggested that
this gene was a major target for this response.
FUTURE PROSPECTS
As GAs are major effectors of plant growth and development,
their biosynthesis is subject to strict regulation throughout most
of the plant growth cycle and in response to environmental
stimuli. Recent research on GA metabolism has been directed
towards understanding regulation, which occurs primarily on
the steps catalysed by ODDs. Many molecular components that
mediate GA metabolic regulation have been identified, although
our understanding of these processes is far from complete. In
particular, the mechanisms underlying GA homoeostasis are
proving surprisingly complex, perhaps at least in part as the
result of the promiscuous interactions of the DELLA proteins,
which mediate this process. Understanding the nature of DELLAmediated signalling and identifying the many partners of these
proteins is currently a rapidly advancing area of research. There
is emerging evidence that DELLAs may act as a point of
convergence for different hormone signalling pathways, thereby
providing a channel by which these pathways may influence GA
metabolism. However, it is clear that auxin signalling and, in
certain circumstances also that of ABA, modify GA biosynthesis
directly without the intervention of DELLAs [132,136]. Details
of these mechanisms at the molecular level should become clearer
in the next few years.
There is also still much to learn about the sites of GA synthesis,
particularly at the cellular level. The increasing evidence that GAs
may act remotely from their source is stimulating renewed interest
in GA movement, which may occur over long distances within
the vasculature, particularly the phloem, or by diffusion between
neighbouring cells. Improved physicochemical and molecular
methods are required to probe GA biosynthesis and action at
greater tissue resolution than is currently possible.
Although GA signalling influences most aspects of plant
development, the specificity of expression domains and
consequently physiological function of the ODD paralogues in
GA metabolism has enabled the identification of GA-deficient
mutants with desirable agricultural traits. For example, the sd1
mutant of rice, lacking OsGA20ox2, and le pea, with a mutant
allele of PsGA3ox1, have reduced stem height, but normal fertility.
Reverse genetic approaches such as TILLING (Targeting Induced
Local Lesions in Genomes) provide an opportunity to introduce
such beneficial mutations into many more agriculturally important
species, although a fuller understanding of the physiological roles
of the target genes is required. The rapidly expanding number of
plant genome sequences available is simplifying the task of gene
identification, allowing suitable targets to be selected for this
purpose. Furthermore, the analysis of natural genetic variation in
these genes between varieties or ecotypes will provide important
information on their influence on plant phenotype. These and
other developments are likely to enhance the already substantial
impact that GA-related research has had on global agriculture.
FUNDING
Rothamsted Research receives strategic support from the Biotechnology and Biological
Sciences Research Council, U.K.
21
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Received 9 February 2012/1 March 2012; accepted 2 March 2012
Published on the Internet 26 April 2012, doi:10.1042/BJ20120245
c The Authors Journal compilation c 2012 Biochemical Society