review article gibberellin biosynthesis and its regulationreview article gibberellin biosynthesis...

Biochem. J. (2012) 444, 11–25 (Printed in Great Britain) doi:10.1042/BJ20120245 11

REVIEW ARTICLEGibberellin biosynthesis and its regulationPeter HEDDEN1 and Stephen G. THOMASRothamsted Research, Harpenden AL5 2JQ, U.K.

The GAs (gibberellins) comprise a large group of diterpenoidcarboxylic acids that are ubiquitous in higher plants, in whichcertain members function as endogenous growth regulators,promoting organ expansion and developmental changes. Thesecompounds are also produced by some species of lower plants,fungi and bacteria, although, in contrast to higher plants, thefunction of GAs in these organisms has only recently beeninvestigated and is still unclear. In higher plants, GAs aresynthesized by the action of terpene cyclases, cytochrome P450mono-oxygenases and 2-oxoglutarate-dependent dioxygenaseslocalized, respectively, in plastids, the endomembrane systemand the cytosol. The concentration of biologically active GAs

at their sites of action is tightly regulated and is moderatedby numerous developmental and environmental cues. Recentresearch has focused on regulatory mechanisms, acting primarilyon expression of the genes that encode the dioxygenases involvedin biosynthesis and deactivation. The present review discussesthe current state of knowledge on GA metabolism with particularemphasis on regulation, including the complex mechanisms forthe maintenance of GA homoeostasis.

Key words: gibberellin biosynthesis, gibberellin deactivation,gibberellin homoeostasis, plant development.

INTRODUCTION

The name GA (gibberellin) encompasses a large group oftetracyclic diterpenoid carboxylic acids with the ent-gibberellane(C20) or 20-nor-ent-gibberellane (C19) carbon skeletons, asexemplified by the simplest members of each class, GA12

and GA9 respectively (Figure 1). Although first identified assecondary metabolites of the fungus Gibberella fujikuroi, theyare ubiquitous in higher plants, in which certain members ofthe group function as endogenous growth regulators. They arealso present in certain species of endophytic bacteria, severalfungal species and some lower plants, but their function inthese organisms is unclear. Currently 136 gibberellin structureshave been identified from all sources and are assigned thetrivial names gibberellin A1–A136, abbreviated to GA1 etc.,numbered in order of discovery [1]. Their structures can befound at http://www.plant-hormones.info/gibberellins.htm. Thereare certainly further GAs still to be discovered, but due to theirvery low abundance, their chemical characterization is far fromtrivial, depending on comparison with chemically synthesizedstandards, so that identification of new GAs has become anincreasingly rare event.

Relatively few GAs have intrinsic biological activity in higherplants, the most common active forms being GA1, GA3 andGA4 (Figure 1). The universal occurrence of GA1 and GA4 inplants suggests that these are the functionally active (hormonal)forms, at least for growth promotion; they co-occur with their

biosynthetic precursors and metabolites, which are often presentat much higher concentrations than the hormones themselves.Gibberellin A3, also known as gibberellic acid, is best knownas the main GA product of G. fujikuroi (now reclassified asFusarium fujikuroi), from which it is produced for commercialapplication, 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, thevast majority of the different GA structures were identified fromimmature seeds, whereas vegetative and early reproductive tissuescontain a relatively restricted and conserved array of structures,representing members of the biosynthetic pathways to GA1 andGA4 and their metabolites. Although there is clear evidence fora regulatory role for GAs in early seed development [2], thefunction of the very high concentrations of active and inactiveGAs present in endosperm, cotyledons and/or testa of seeds at laterdevelopmental stages is unclear. Seeds approaching maturity oftencontain high levels of GA-inactivating activity, ensuring againstconcentrations of bioactive GAs in mature seed that could provokepremature germination and abnormal seedling growth [3].

Physiologically, a major function of GAs in higher plants can begeneralized as stimulating organ growth through enhancement ofcell elongation and, in some cases, cell division. In addition, GAspromote certain developmental switches, such as between seeddormancy 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

Abbreviations used: ABA, abscisic acid; AG, AGAMOUS; AGF1, AT-hook protein of GA feedback 1; bHLH, basic helix–loop–helix; bZIP, basic leucinezipper; CBF, C-repeat-binding factor; CHO1, CHOTTO1; CPS, ent-copalyl diphosphate synthase; DAG1, DOF AFFECTING GERMINATION 1; DDF, DWARFAND DELAYED FLOWERING 1; DIF, difference between day temperature and night temperature; DOG1, DELAY OF GERMINATION 1; DRE, dehydration-response element; DREB, DRE-binding; EUI, ELONGATED UPPERMOST INTERNODE; FUS3, FUSCA3; GA, gibberellin; GA2ox, GA 2-oxidase; GA3ox, GA3-oxidase; GA20ox, GA 20-oxidase; GAMT, GA methyl transferase; GGPP, trans-geranylgeranyl diphosphate; GGPS, trans-geranylgeranyl diphosphatesynthase; 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 (minichromosomemaintenance1), AGAMOUS, DEFICIENS and SRF (serum-response factor); LD, long day; LEC, LEAFY COTYLEDON; MS, male-sterile; ODD, 2-oxoglutarate-dependent 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, SCARECROW-LIKE 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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http://www.plant-hormones.info/gibberellins.htm

12 P. Hedden and S. G. Thomas

Figure 1 Structures of selected GAs

Included are the simplest example of a C20-GA, GA12, with the carbon numbering system, andthe 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, inaddition to allowing stamen elongation, they are necessary for thedevelopment, release and germination of pollen and for pollentube growth. In carrying out their functions GAs act in responseto developmental and environmental cues, which may regulateGA biosynthesis, turnover (deactivation), perception or signaltransduction, sometimes acting at several points in the pathway.

Impressive progress has been made within the last decade inestablishing details of the GA biosynthesis and signal transductionpathways. The advances in our understanding of the GAsignal transduction cascade (reviewed in [4–7]) have stemmedparticularly from the discovery that GID (GIBBERELLIN-INSENSITIVE DWARF) 1 encodes a soluble GA receptor in rice[8]. A pathway has emerged in which growth repression is exertedby DELLA proteins through their regulation of transcription([9]; also reviewed in [4]). In response to environmental anddevelopmental stimuli, this repression is relieved through theproduction of biologically active GAs, which rapidly promoteDELLA degradation. Gibberellins bind within a pocket of GID1,causing a conformational change that facilitates the formation ofa GA–GID1–DELLA complex and the subsequent ubiquitinationof DELLAs and their destruction by the 26S proteasome ([10–12];also reviewed in [7]). In the absence of a functional degradationpathway, binding of GA–GID1 has also been demonstrated to havea repressive effect on DELLA function, although the physiologicalsignificance has yet to be established [13,14]. There is currentlylittle evidence that DELLAs act as transcription factors thatdirectly control the expression of GA response genes. However,recent studies demonstrated that DELLA repression of hypocotylexpansion is mediated through their binding and sequestrationof the PIF (PHYTOCHROME-INTERACTING FACTOR) 3 andPIF4 bHLH (basic helix–loop–helix) transcription factors [15,16].There is now strong evidence that DELLAs also regulate theactivity of other classes of transcriptional regulators, includingthe JAZs (JASMONATE ZIM-domain proteins). In this case,DELLAs were shown to modulate jasmonate signalling bybinding to JAZs and thereby blocking their inhibitory effect ondownstream transcriptional events [17].

The present review focuses on GA biosynthesis anddeactivation: after a discussion of the pathways, we will highlightrecent advances in our understanding of how they are regulated.

THE GA BIOSYNTHETIC PATHWAY

This topic was reviewed previously by Yamaguchi [18]. In higherplants, GAs are formed primarily from the methylerythritolphosphate pathway [19], by which the hydrocarbon intermediateent-kaurene is produced from GGPP (trans-geranylgeranyldiphosphate) in proplastids [20]. The biosynthetic pathway fromGGPP to the bioactive GAs is presented in Figure 2. It has beengenerally assumed that ent-kaurene synthesis competes with thatof other GGPP-derived metabolites for a common pool of GGPP(e.g. [21]). However, the finding that loss of geranyl diphosphatesynthase in tomato and Arabidopsis thaliana (hereafter referredto as Arabidopsis) reduces GA content without affecting thelevels of carotenoids and chlorophylls suggests that ent-kaureneis produced from a separate pool of GGPP, via a GGPS (GGPPsynthase) that, in contrast with the GGPS isozymes involved inpigment synthesis, has a requirement for GPP as substrate [22].This would require that GAs and the GGPP-derived pigments areproduced in different tissues and is consistent with ent-kaurenesynthesis in leaves occurring in immature chloroplasts associatedwith the vasculature [23]. Plants contain relatively many GGPSgenes, the Arabidopsis genome containing 12 such genes, withmost of the encoded enzymes localized in plastids [24], and itis feasible that one or more of these may be dedicated to GAbiosynthesis. The fungus F. fujikuroi contains two GGPS genes,one of which is located in the GA biosynthesis gene cluster andis used exclusively for GA biosynthesis [25].

Synthesis of ent-kaurene from GGPP occurs in two stepsvia ent-copalyl diphosphate, the reactions catalysed by separateenzymes, 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 higherplants [26]. These reactions are also catalysed by separate CPSand KS enzymes in the GA-producing bacterium Bradyrhizobiumjaponicum [27], whereas in fungi both activities reside in a singlepolypeptide [28]. In Arabidopsis, both CPS and KS are encodedby single genes, loss of function of which results in severe GA-deficient phenotypes, characterized by extreme dwarfism, maleand female infertility and non-germinating seeds [29]. Severalspecies, particularly cereals, have been found to express two ormore CPS- and KS-like genes [30–33], although in rice only onemember of each gene family is thought to be involved in GAbiosynthesis, the others producing phytoalexins [30,33].

ent-Kaurene is relatively volatile and has been foundto exchange with the external environment, prompting thesuggestion that it may function as a mediator of plant–plant communication [34]. However, since regulation of GAbiosynthesis occurs mainly at later stages of the pathway [35],exogenous ent-kaurene is unlikely to have a major influence onGA content and the resulting growth.

The conversion of ent-kaurene into the bioactive forms involvesthe action of membrane-associated P450s (cytochrome P450mono-oxygenases) and soluble ODDs (2-oxoglutarate-dependentdioxygenases). The formation of GA12, which is consideredthe common precursor for all GAs in plants [36], requires sixoxidative steps, catalysed by two mono-oxygenases, KO (ent-kaurene oxidase) and KAO (ent-kaurenoic acid oxidase). KObelongs to the CYP701A P450 clade and KAO to CYP88A [37].These enzymes are localized in the endoplasmic reticulum and, inthe case of KO, also in the plastid envelope [38]. The three-stepKO-catalysed oxidation of ent-kaurene to ent-kaurenoic acid viaent-kaurenol and ent-kaurenal has been shown to involve repeatedhydroxylations on C-19, with the initial step rate-limiting andthe intermediates retained at the enzyme active site [39]. KO isencoded by a single gene in Arabidopsis, whereas rice contains


Regulation of gibberellin biosynthesis 13

Figure 2 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 theenzymes 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 KOactivity [40]. Mutations in OsKO2 cause severe GA-deficiencyand dwarfism, confirming its involvement in GA-biosynthesis andsuggesting it may be the only gene of the cluster to have thisrole [30].

The oxidation of ent-kaurenoic acid to GA12 by KAO occursin three steps via the intermediates ent-7α-hydroxykaurenoicacid and GA12-aldehyde, requiring successive oxidations at C-7β, C-6β and C-7 (evidence summarized in [41]). In fungiand developing seeds of some plant species in which GAsare produced at very high levels, the first two of thesereactions produce by-products (kaurenolides and fujenoic acidsrespectively) in substantial quantities, but these do not appearto be formed in tissues in which GAs are produced in hormonalquantities. The fungus F. fujikuroi also employs cytochrome P450sfor the KO and KAO reactions, but they are unrelated to the higherplant enzymes, belonging to the CYP503 and CYP68 cladesrespectively, and appear to have evolved independently [42]. TheF. fujikuroi KAO produces mainly GA14 (3β-hydroxyGA12) ratherthan GA12 due to 3β-hydroxylation of GA12-aldehyde, an activitynot present in the higher plant enzymes [43].

Whereas rice contains a single KAO gene [30], Arabidopsiscontains two fully redundant copies [44]. Two KAO genes havealso been reported in pea and sunflower, but these genes differ

in expression domains, with one gene being expressed in mosttissues, whereas expression of the second gene is restricted todeveloping seeds and roots [45,46]. The pea enzymes were foundto produce small amounts of the non-GA by-products in vitro [45],perhaps in response to high substrate concentrations. Lesions inKAO have been shown to be the cause of severe dwarfism in severalspecies, including rice (d35 mutant) [30], maize (dwarf3) [44], pea(na) [45] and sunflower (dwarf2) [46]. Pumpkin contains an ODDthat oxidizes GA12-aldehyde to GA12 [47], but orthologues of thisenzyme 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 theenzymatic activity responsible for 13-hydroxylation, by whichGA12 is converted into GA53, is unclear. Both P450 and ODD13-hydroxylase activities have been detected (reviewed in [48]),and it has been recently reported that rice contains two P450s thatconvert GA12 into GA53 [49]. Interestingly, the enzymes presentin 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 (GA20-oxidase), an ODD, converting these substrates in parallelpathways into GA9 and GA20 respectively. This is a three- orfour-step process, depending on the mechanism for the loss ofC-20, which has not been determined [41]. In this reaction series,the C-20 methyl group is oxidized to the alcohol and then the



aldehyde, from which it is lost with the formation of a γ -lactonebetween C-19 and C-10. The alcohol and aldehyde intermediatesaccumulate in plants, the aldehydes often to relatively high levels[52], consistent with them being released and having to rebind tothe enzyme active site for the successive oxidations. In contrastwith the accumulation of these intermediates, the intermediates inthe P450-catalysed reactions earlier in the pathway are normallypresent in only trace quantities [53]. The C-20 alcohol readilylactonizes with the C-19 carboxylic acid group, particularlyat low pH, preventing further oxidation by the GA20ox [54].Moreover, some tissues contain a 20-oxidase activity capableof oxidizing C-20 in its lactone form [54], although there islittle information on the nature of the enzyme responsible forthis activity. It is not known to what extent lactonization ofC-20 alcohols occurs in planta, because the acidic conditions usedduring GA analysis promotes lactone formation, but the existenceof the lactone-oxidizing activity suggests that these δ-lactonesmay 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 onC-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] necessitatesthe existence of an intermediate between the C20 aldehyde and theC19 γ -lactone product. However, no candidate intermediate hasbeen identified and it has been suggested that it remains boundat the enzyme active site [41]. A side reaction from the oxidationof the aldehyde results in the formation of a carboxylic acid.This reaction can occur from a substantial to a negligible extentdepending on the nature of the GA20ox, and is most commonin developing seeds (see, e.g., [57]). The resulting tricarboxylicacid product is not converted into C19-GAs and constitutes a bio-logically inactive end-product. Oxidative loss of C-20 in the fungiF. fujikuroi and Sphaceloma manihoticola is catalysed by a P450rather than an ODD [58,59]. Apart from GA14, the first substratefor the fungal enzyme, the proposed C20-GA intermediates (GA37

and GA36) in the formation of GA4 accumulate to a much smallerextent in fungal cultures than do the equivalent intermediatesin plants, and, in contrast with the plant intermediates, are notoxidized to C19-GA products when added to fungal cultures [59].This may indicate that these fungal intermediates do not leavethe active site, and their presence in fungal cultures may be dueto hydrolysis of enzyme-bound intermediates [41]. However,cultures of F. fujikuroi grown in the presence of 18O2 or H2

18Oincorporated 18O only from 18O2 into the intermediates, indicatingthat they were not formed by hydrolysis [60].

The final step in the formation of the biologically activehormones is the 3β-hydroxylation of GA9 and GA20 to GA4

and GA1 respectively, catalysed by the ODD GA3ox (GA 3-oxidase). In shoots of dicotyledonous species this reaction tendsto be highly regiospecific, with a single product being produced[61–63], whereas in several monocotyledons, in addition toGA1, GA3 is formed from GA20 via the intermediate GA5 bythe action of a single GA3ox [64–66]. Developing seeds ofcertain dicotyledonous species contain GA3oxs of relatively lowregiospecificity, possessing 2β-hydroxylase [65,67,68] and/or2,3-desaturase [65,69,70] activities in addition to their 3β-hydroxylase activity. C-2,3-unsaturated GAs may be furtheroxidized by GA3oxs to the 2,3-epoxide or to the 3β-hydroxy-1,2-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 andthen addition of OH at C-3β [69]. Whereas both desaturationand formation of the 3β-hydroxy-1,2-unsaturated productare catalysed by a single enzyme in cereals, separate enzymes arerequired for these reactions in developing Marah macrocarpus

seeds [70]. In an example of extreme lack of regiospecificity, twoGA3ox enzymes, from wheat [64] and M. macrocarpus seeds[70], were shown to possess some 13-hydroxylase activity. Thisrequires that the substrate binds to the enzyme active site inopposite orientations to be oxidized either on C-3 or C-13, andindeed C19-GA substrates, such as GA9, possess some symmetry,with the 6-carboxylic and 19,10-lactone moieties potentially ableto exchange positions in the binding site. Although the 13-hydroxylase activity of the M. macrocarpus enzyme may havesome significance in seeds of this species, in which low amountsof 13-hydroxy-C19-GA, but no 13-hydroxy-C20-GAs, are present,consistent with this reaction occurring late in the pathway, early13-hydroxylation by a regiospecific enzyme appears to be themost common route in higher plants. In the fungus F. fujikuroi,13-hydroxylation of GA7 to GA3 by a P450 is the final step in thebiosynthesis of GA3 in this species [71].

In contrast with the earlier enzymes, the ODDs are encodedby multiple genes in all plant species that have been studied:for example there are five GA20ox enzymes in Arabidopsis andfour in rice, whereas these species contain four and two GA3oxgenes respectively [72]. Members of these gene families varyconsiderably in their levels and patterns of expression, and aredifferentially regulated (see below). This multiplicity of genesmay be related to the fact that the GA biosynthetic pathway isregulated primarily by the activity of the ODDs.

GIBBERELLIN DEACTIVATION

It is essential for plants to be able to regulate precisely their GAcontent and to have the ability to change this parameter rapidly inresponse to changes in their environment. In common with otherplant hormones, GAs are inactivated and this provides a meansto regulate homoeostasis and to allow a rapid reduction of GAconcentration when required. Several mechanisms for inactivatingGAs have been identified (Figure 3), the most prevalent being 2β-hydroxylation. The enzymes responsible for this activity, GA2oxs(GA 2-oxidases), are ODDs, which can be divided into twomajor groups according to function: one group acts on C19-GAs,including the bioactive compounds and their immediate non-3β-hydroxylated precursors [73], whereas another group acts on C20-GAs [74]. The C19-GA2oxs can be divided into two subgroups onthe basis of amino acid sequence [75–77] and it has been proposedthat they differ also in biochemical function [76]. Several C19-GA2oxs, all of which belong to the same subgroup, have beenshown to be bifunctional, acting as 2β-hydroxylases, but alsofurther oxidizing the 2β-hydroxy group to a ketone. The isolated2-keto products, known as GA-catabolites [78], are dicarboxylicacids with a double bond at C-1,10, C-5,10 or C-10,11 (shown forthe �1,10 isomer in Figure 3) that are thought to arise from the 2-ketone 19,10-lactones by rearrangement, probably during analysis[42]. The catabolites can accumulate to high concentrationsin seeds of some species, for example, pea [79]. The secondsubgroup of C19-GA2oxs do not appear to possess the capacityto produce the GA-catabolites [76]. The GA2ox enzymes formparticularly large gene families, although, on the basis of sequencephylogeny, the C20-GA2oxs might be considered a separate familyfrom those acting on C19-GAs [80]. In common with the otherODDs, the genes encoding GA2oxs are major sites of regulation(see below).

Other mechanisms for GA deactivation have been recognized inrecent years. A cytochrome P450 mono-oxygenase (CYP714D1),which is encoded by the EUI (ELONGATED UPPERMOSTINTERNODE) gene in rice, was shown in vitro to convert GAsinto their 16α,17-epoxides [81]. The enzyme accepted GA12,



Figure 3 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 theepoxidase EUI (green arrows). Gibberellin methyl esters are formed in developing seeds of Arabidopsis by SAMT methyl transferases (blue arrows). The brown arrows indicate reversible formationof GA glucosyl esters. The functionality introduced in each case is indicated by the appropriate colour.

GA9 and GA4 as substrates (other 13-desoxyGAs were nottested), but was much less effective with 13-hydroxylated GAs.Overexpression of EUI in rice caused extreme dwarfism and areduction in GA4 content in the uppermost internode, confirmingthat epoxidation resulted in GA deactivation. However, it wasnot possible to identify the epoxides in plant tissues, evenwhen EUI was overexpressed, but the accumulation of GA-16α,17-dihydrodiols indicates that the epoxides are hydratedin planta, either enzymatically or by a non-enzymatic acid-catalysed reaction. GA dihydrodiols have been found to occurnaturally in many plant species (discussed in [81]), suggestingthat 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, withCYP714A1 producing the most severe effect [82], although thefunction 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 failureof the panicle to emerge fully from the leaf sheaths, a syndromethat was effectively overcome by the extended upper internodesresulting from the presence of the eui mutation. The effect of thismutation is consistent with the expression pattern of EUI, which,on the basis of reporter gene analysis, occurs most strongly in theflowering spikelets and in the nodes and cell division zone ofthe expanding internodes [81]. Rice anthers produce extremelyhigh levels of GA4 [84], and it is possible that EUI may regulatethe influx of GA4 into the stem from reproductive tissues, whereasGA1 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, presentin Arabidopsis, were shown to be specific for GAs [86,87]. Theenzymes methylate the 6-carboxy group of C19-GAs, a process thatabolishes biological activity [88]. Thus ectopic overexpression ofthese genes in Arabidopsis resulted in dwarfism. The GAMT genesare expressed in developing seeds, where they apparently function

to regulate GA content, since gene knockout lines accumulatedGAs [86]. Their physiological roles may be restricted to seeddevelopment and the control of precocious germination.

GIBBERELLIN CONJUGATES

Ester and ether GA conjugates have been identified in a range ofplant species, with glucose the predominant conjugate partner[89]. Although there has been interest in these compoundsas potential storage and/or transport forms of GA, it wasdifficult to assign to them a definite physiological role, andattention to this topic has waned in recent years. Consequently,there is limited information on the enzymes involved in theirformation or hydrolysis. Some GA conjugates were shownto have biological activity in bioassays, but this was thought tobe due to hydrolysis to the aglycone in the assay system [90].Evidence for the reversibility of GA conjugate formation addsto the feasibility of their having a storage role [91], providinga mechanism for rapid release of bioactive GA that would,for example, stimulate seed germination. A recent report thatectopic expression of a β-glucosidase in transplastomic tobaccoresulted in increased levels of several hormones including GA1

and GA4 [92] suggests that plastids may act as a store ofhormone conjugates. Conjugation might also provide a potentiallyreversible deactivation mechanism, although evidence for thisis lacking. It is of interest that 2β-hydroxyGAs form glucosylethers linked through the 2β-hydroxyl group [89]. In this case,conjugation may serve to solubilize and trap these inactive end-products in the vacuole.

REGULATION OF GA BIOSYNTHESIS

Sites of synthesis

In general, the highest levels of bioactive GAs are found in activelygrowing organs, for example developing leaves and expandinginternodes [93]. Such organs contain high expression levels of



GA-biosynthetic genes, indicating active GA synthesis [23,94].On the basis of the coincidence of expression of GA20ox andGA3ox 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 suchorgans. There are several examples of different reactions of thepathway occurring in separate tissues, requiring the movementof 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 expressedin the provasculature of the embryonic axis in germinatingArabidopsis seeds, KO and GA3ox genes were expressed in thecortex and endodermis. This would require the movement of anintermediate, potentially ent-kaurene, from the provasculature, ahighly plausible scenario given the demonstrated release of thisintermediate into the environment [34]. The synthesis of ent-kaurene from GGPP can be considered the gateway into the GAbiosynthetic pathway and as such would control the metabolicflux into the pathway [23]. On the basis of incubations with cell-free systems from wheat tissues, Aach et al. [20] concluded thatent-kaurene is synthesized in proplastids within regions of celldivision, but not in fully developed chloroplasts in elongating ormature cells. In the stem, they found the highest activity in thenode, assumed to be associated with the intercalary meristem.This region was also reported to be the major site of TaGA20ox1expression in the upper wheat stem, with TaGA3ox1 expressiondivided 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 ofGAs or precursors from the meristem to the elongation zone, oralternatively GAs and intermediates may remain in the dividingcells as they move into the elongation zone. In contrast with thisresult, Kaneko et al. [94] reported promoter–GUS expression forthe 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 bySilverstone et al. [98] in the vasculature of mature leaves, in whichthe chloroplasts are immature and may thus be still capable ofent-kaurene synthesis. Indeed, Ross et al. [99] demonstrated thatmature pea leaves and internodes were active for GA biosynthesis,but contained low levels of GA1 and GA20 due to high rates of2β-hydroxylation. The potential association of GA biosynthesiswith the vasculature raises the possibility that this tissue mayact as a source of GAs for export via the phloem. There is infact considerable evidence from experiments involving graftingand application of labelled GAs for long-distance movement ofGAs in the vasculature (reviewed in [100]). Results from suchexperiments with pea [101] indicated that GA20 rather than GA1

or earlier intermediates is the mobile form in this species. It hasbeen demonstrated in a number of species that leaves produceGAs 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 exportedfrom the leaves [102], whereas in Lolium species, the mobilesignal, produced in the leaves in response to long days, wassuggested to be GA5 and GA6, which would escape metabolism byGA2oxs [103]. A discussion of the regulation of GA biosynthesisby photoperiod is given below.

Certain tissues, which are particularly active in GAbiosynthesis, function as sources of GA to support thedevelopment of neighbouring tissues. In germinating cerealgrains, the scutellum epithelium exports GAs to the non-GA-producing aleurone cells [94], which respond by synthesizingand secreting hydrolases to the endosperm as well as undergoingPCD (programmed cell death) [104]. Within flowers, the highest

concentrations of GAs occur in anthers, in which the tapetumhas been identified as an important site of synthesis [84,105].Interestingly, the tapetum also undergoes PCD in response toGA, to release components that are incorporated into the pollenwall [106]. Some flower organs that are non-autonomous forGAs, particularly the petals, are dependent on the anthers,and potentially also the receptacle, to supply these hormonesfor normal development [105,107]. The suspensor, an organpresent in the seed during early embryogenesis that supportsgrowth of the young embryo [108], has been shown in severalspecies to be an extremely rich source of GAs, which arethought to be important for early embryo growth (reviewedin [109]). Later in development, the immature seed of many,but not all, species contain high levels of GAs, which maybe produced in the endosperm and/or the cotyledons. Theseorgans can produce a vast range of different GA structuresnot found in other tissues [110], the function of which isunclear. A characteristic of these high-GA-producing tissues is therecruitment of several ODD paralogues. For example, in the ricescutellum and tapetum, OsGA20ox1 and OsGA3ox1 are expressedin addition to OsGA20ox2 and OsGA3ox2, the first two genesnot being expressed elsewhere [94]. Tissues of developing seedstypically express genes not active in other tissues, as for examplefound in pumpkin [111]. These genes are presumably not subjectto the regulatory constraints imposed by the normal mechanismfor GA homoeostasis (see below).

Developmental regulation

Consistent with its proposed role as the ‘gatekeeper’ for GAbiosynthesis, CPS expression is highly developmentally regulatedas noted above [23]. Although CPS activity may regulatethe flux through the GA biosynthetic pathway, the activityof the ODDs more precisely determines the concentration ofbioactive GA [35]. The different ODD paralogous genes typicallyshow distinct, but overlapping, expression patterns and as suchcontribute unequally to different developmental processes. Forexample, in Arabidopsis, loss of AtGA20ox1, but of no otherindividual GA20ox, results in a reduction in stem internode length,whereas additional loss of AtGA20ox2 causes further internodeshortening, indicating that the latter also contributes to inter-node length [112]. However, this contribution is exaggerated inthe absence of AtGA20ox1, due to compensatory mechanisms(see below). A similar relationship exists between AtGA3ox1and AtGA3ox2 in the regulation of stem extension [113].

Some indications of the molecular mechanisms underlyingthe developmental regulation of GA biosynthesis have beenreported. The LEC (LEAFY COTYLEDON) transcription factorsLEC2 and FUS3 (FUSCA3), which are required for correcttissue specification during embryogenesis and for normal seeddevelopment, suppress GA biosynthesis in the developing embryoby down-regulating AtGA3ox2 expression in the epidermis [114].FUS3 was shown to bind to RY elements in the AtGA3ox2promoter. The requirement to restrict GA production duringembryogenesis is reinforced by the findings of Wang et al.[115], who reported that the GA-deactivation gene AtGA2ox6 wasup-regulated in the developing embryo by the MADS [MCM1(minichromosome maintenance 1), AGAMOUS, DEFICIENSand SRF (serum-response factor)] domain transcription factorAGAMOUS-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 thisstage of development [114]. Another MADS domain proteinAG (AGAMOUS) was shown to directly promote expressionof AtGA3ox1 in developing flowers [116]. AG function is



necessary for correct floral organ specification, but also for theirdevelopment, and although organ specification can occur in theabsence of GA signalling [117], GAs are necessary for floral organdevelopment [118]. AtGA3ox1 was also shown to be a direct targetof the bHLH transcription factor INDETERMINATE in the valvemargins and septum of the Arabidopsis silique, as part of theprocess of pod opening and seed dispersal [119].

In the vegetative shoot apex, GA biosynthesis occurs in leafprimordia and in the elongating sub-apical region [120]. However,in order to maintain a pool of indeterminant cells in the shootapical meristem, it is proposed that bioactive GAs must beexcluded from this region through the action of homeodomaintranscription factors of the Class-I KNOX (KNOTTED-likehomeobox) class, which suppress the expression of GA20ox genes[120,121]. The tobacco homeobox NTH15 (Nicotiana tabacumhomeobox 15) was shown to interact with a sequence within thefirst intron of the NtGA20ox1 gene [121]. In potato, suppression ofStGA20ox1 expression was mediated by both the KNOX proteinPOTH1 and a BEL-type homeodomain protein, StBEL5, whichbound as a heterodimer to the promoter [122]. The expression ofGA2ox genes has been localized to the base of the meristemin several species where the enzyme may function to protectthe meristem from the influx of GAs [123–126]. Expressionof the rice gene OsGA2ox1 is reduced when plants are grownin florally inductive short days, so permitting an influx of GAsfor promotion of the floral transition [125]. In Arabidopsis,expression of AtGA2ox2 and AtGA2ox4 was up-regulated byinduction of the KNOX protein SHOOTMERISTEMLESS andby cytokinin, which was proposed to mediate this effect throughKNOX-induced expression of the cytokinin-biosynthetic geneISOPENTENYLTRANSFERASE [124]. However, direct inductionof GA2ox expression by KNOX was demonstrated in maize, withthe homeodomain protein KNOTTED1 binding to the first intronof ZmGA2ox1 [123].

Regulation of GA biosynthesis by other hormones

There has been considerable interest in recent years in interactionsbetween hormone signalling pathways [127]. Such interactionsare consistent with the overlapping physiological roles found forthe different hormones, although this is not so clearly reflectedat the molecular level, with, in some cases, the hormonesregulating different, albeit functionally related, genes [128].Interactions through regulation of hormone biosynthesis havebeen invoked in a number of cases, although direct evidence forthis is rarely presented and its occurrence may be exaggerated[129]. However, there is strong evidence for regulation of GAconcentration by auxin signalling at the level of ODD geneexpression, with up-regulation of GA20ox and/or GA3ox genesand, in some cases, down-regulation of GA2ox genes resultingin increased GA content [130–134]. The molecular mechanismfor auxin regulation of GA-biosynthesis gene expression is notknown, but appears to be direct and does not involve DELLAproteins [130,132], although the effect may be masked byDELLA-mediated homoeostasis in some cases [135].

Although Ross et al. [129] could find no evidence for regulationof GA metabolism by ABA (abscisic acid) in pea internodes, aninteraction between these hormones has been shown in the contextof seed germination [136]. The CHO1 (CHOTTO1) transcriptionfactor, which enhances dormancy in imbibed Arabidopsis seeds,inhibits GA biosynthesis through suppression of AtGA3ox2expression [136]. Since expression of CHO1 requires ABAsignalling [137], this transcription factor couples the regulation ofGA biosynthesis to ABA signalling. There has been no evidence

that CHO1 binds directly to the AtGA3ox2 promoter, so theregulation may be indirect or involve interaction with othertranscription factors, either scenario being consistent with thisregulation being specific to imbibed seeds despite expression ofCHO1 occurring in other organs [136].

Gibberellin homoeostasis

Even prior to the identification of genes encoding GA metabolicenzymes or GA signalling components, it was apparent thathomoeostatic mechanism(s) existed to maintain optimal GA levelsfor co-ordinated plant growth and development. The presentreview will focus on recent advances in understanding themolecular events involved in this mechanism, summarized inFigure 4. However, it is important to acknowledge that it ispart of a wider homoeostatic mechanism, also including theregulation of transcription of core GA signalling components[13,14,112,117,138,139], which potentially co-ordinates GA-responsive growth through maintenance of optimal levels of thegrowth-repressing DELLA proteins.

The biochemical study of GA biosynthetic and responsemutants in several plant species demonstrated that GA levels arecontrolled through a process of feedback regulation (reviewedin [140]). The subsequent cloning of genes encoding GAbiosynthesis enzymes led to the demonstration that this regulationwas mediated through the transcriptional control of specificODDs. For example, in Arabidopsis this includes three GA20oxgenes (GA20ox1, GA20ox2 and GA20ox3) and a single GA3ox(GA3ox1) whose expression levels are all highly elevated whenbioactive GA levels are low, but are then rapidly reduced whenthese plants are treated with exogenous GA [112,113,141,142].In contrast, other members of these two gene families, in additionto those encoding enzymes catalysing all of the earlier steps in theGA biosynthetic pathway, do not appear to be subject to feedbackregulation [112,113,143]. An additional level of regulation thatfunctions in GA homoeostasis involves the feedforward regulationof GA2ox genes [73,75,138]. Studies in Arabidopsis havedemonstrated that multiple members of both the C19 and C20

classes of GA2oxs are transcriptionally up-regulated in responseto increased GA signalling output [73,138,144]. On the basis ofthese observations it is likely that GA-mediated transcriptionalchanges in the ODD genes translate to alterations in the levelsof the metabolic enzymes they encode, ultimately leading to thechanges in flux of GAs through this pathway that are necessaryfor controlling the levels of bioactive GAs. The low abundanceand labile nature of these metabolic enzymes has hindered theirdetection and the subsequent confirmation of this hypothesis. Anexception to this was a recent study of the photoperiodic regulationof a GA20ox gene in spinach, in which the authors were able todetect the endogenous SoGA20ox1 protein by Western blotting[145]. Although they found no evidence for the transcriptionalfeedback regulation of this gene in petioles and shoot tips ofplants treated with GA biosynthesis inhibitors, these treatmentsresulted in a clear increase in SoGA20ox1 protein levels. Thesefindings allude to further complexities in GA homoeostasisthrough potential post-transcriptional regulatory mechanisms.In light of the importance of targeted protein degradation inregulating GA signal transduction, it is tempting to speculatethat similar mechanisms may exist to control the regulation ofGA metabolism. A precedence for this is the feedback regulationof ACC synthase stability by ethylene signalling [146].

Recent advances in our understanding of GA signaltransduction have provided important insights into themechanisms controlling GA homoeostasis. There is conclusiveevidence demonstrating that transcriptional feedback and



Figure 4 Homoeostatic regulation of GA metabolism and signalling

In the absence of GA signalling, DELLAs increase GA levels through transcriptional control of GAmetabolic genes. The subsequent increase in GA concentration leads to GID1-mediated DELLAdegradation, allowing GA homoeostasis. The homoeostatic control mechanism also includesthe transcriptional regulation of GA signalling genes by DELLAs. SCL3 acts to attenuate DELLAactivity through direct association. The shaded box indicates transcription factors that have apotential role in controlling the transcriptional feedback regulation of specific GA biosynthesisgenes. Their relationship to the regulation of these genes by DELLAs is currently unclear. Greylines indicate transcriptional control.

feedforward regulation of GA metabolism is mediated by theGA signalling pathway and is dependent on the levels ofDELLA proteins. In the case of feedback regulation, this hasbeen clearly illustrated in mutants lacking core GA signallingcomponents including GID1 [8,117,147,148] and GID2/SLY1(SLEEPY1) [149,150], which are necessary for targeting DELLAdegradation in response to GA. In these mutants, the expressionlevels of feedback-regulated GA biosynthetic genes are highlyelevated and are not down-regulated by exogenous GA treatment.Consequently, these mutants accumulate highly elevated levelsof bioactive GAs [8,117,147,150]. The absolute requirementfor DELLAs in controlling the feedback response has alsobeen demonstrated extensively in many higher plant speciesby characterizing both loss-of-function and gain-of-function (inwhich the abnormal DELLAs are resistant to GA-mediateddegradation) mutants. Notably, it was the study of DELLAgain-of-function mutants in maize, wheat and Arabidopsis thatinitially provided the evidence for feedback regulation of GAbiosynthesis on the basis of the observations that they containedelevated levels of bioactive GAs [151–153]. Subsequently, it hasbeen demonstrated that the transcriptional feedback regulation isperturbed in both classes of DELLA mutants, with transcript levelsfound to be low in those lacking DELLAs, but highly elevated inthose containing stabilized forms [144,154–157]. Most of thesestudies have focused on the role of DELLAs in the feedbackregulation of GA20ox and GA3ox. However, a recent study of aDELLA loss-of-function mutant in pea (la cry) has demonstratedthat the GA-mediated feedforward regulation of PsGA2ox1 andPsGA2ox2 is also controlled by these DELLAs [144].

DELLAs are nuclear-localized proteins that function astranscriptional regulators [9,156,158,159]. Because they actas growth repressors, there is occasionally the misconceivedperception that they are transcriptional repressors. In fact, anearly study of the rice DELLA SLR1 suggested that it actsas a transcriptional activator in yeast [159a]. This was laterconfirmed in a detailed transcriptomics analysis of DELLAtarget genes in Arabidopsis, showing that the majority of

these are up-regulated by DELLA (and down-regulated by GA)[9]. Interestingly, those genes showing the largest changes inexpression in response to alterations in DELLA levels includedthe feedback-regulated genes GA20ox2 and GA3ox1. This workand an earlier study by Gubler et al. [160] demonstrated thatDELLA protein degradation occurs within 5–10 min followingGA treatment of GA-deficient Arabidopsis seedlings and barleyaleurone layers [9,160]. In Arabidopsis, the subsequent reductionsin the transcript levels of GA3ox1 and GA20ox2 were detectableafter 15 min, suggesting that these are probably DELLA primarytarget genes [9]. However, Zentella et al. [9] were unable to detectbinding of an RGA (REPRESSOR OF ga1)–TAP (tandem affinitypurification)-tag fusion protein to the promoters of GA3ox1 orGA20ox2, suggesting that other factors are necessary to regulatetranscription.

There has been considerable interest in attempting tounderstand the mechanisms and identify the transcription factorsthat are responsible for controlling the feedback regulation of GAbiosynthesis. Despite these efforts the picture is still rather unclear,although several transcription factors that appear to have a rolein 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 leucinezipper)-containing transcription factor identified in tobacco [162].The characterization of a dominant-negative form of RSG initiallydemonstrated that it acts as a transcriptional activator of NtKO.It was subsequently demonstrated that RSG also binds to andactivates the expression of the feedback-regulated NtGA20ox1gene [163]. Binding of RSG to the NtGA20ox1 promoter wasfound to be rapidly suppressed by GA, indicating that it mightcontrol the transcriptional feedback regulation of this gene.Interestingly, there was no evidence that RSG controlled the GA-mediated feedback regulation of another GA biosynthetic gene,NtGA3ox [164].

The identification of RSG protein interactors has led tosome important findings about the regulatory mechanismscontrolling RSG transcriptional activity. These comprehensivestudies have demonstrated that 14-3-3 proteins bind to RSG,sequestering it in a cytoplasmic localization in response to GAsignalling and preventing its activation of NtGA20ox1 and othertarget genes [164,165]. This process is controlled by CDPK1(Ca2 + -dependent protein kinase 1), which in response to GAsignalling, and potentially an elevation in intracellular Ca2 +

levels, phosphorylates RSG on Ser114, enhancing its associationwith 14-3-3 proteins in the cytoplasm [164,166]. Althoughthese studies provided important insights into the control offeedback regulation of NtGA20ox1, there are still many remainingquestions. Notably, the role of DELLAs in the regulation ofRSG activity is unclear. It is conceivable that DELLA signallingregulates 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 regulationof GA biosynthesis has not been reported for other plant species,but if this turns out to be the case, the availability of improvedgenetic resources in model species should provide important toolsfor dissecting the mechanisms involved.

In Arabidopsis, characterization of the cis-acting sequencesresponsible for controlling the feedback regulation of AtGA3ox1has led to the identification of AGF1 (AT-hook protein of GAfeedback 1) as a potential regulator of this response [168]. Thepresence of an AT-hook DNA-binding motif together with thedemonstration that an AGF1–GFP (green fluorescent protein)fusion protein is nuclear-localized in transgenic plants supports arole of AGF1 as a transcriptional regulator. However, although theoverexpression of AGF1 potentiates the transcriptional feedback



regulation of AtGA3ox1, a clear role in regulating this responsehas yet to be established.

Members of the YABBY family of C2C2 zinc fingertranscription factors have a well characterized role in themaintenance of polar outgrowth of lateral organs [169]. Althoughextensive studies of the YABBY genes in Arabidopsis have notyet indicated involvement in the regulation of GA metabolism,there is evidence in rice implicating OsYAB1 (OsYABBY1) inthe feedback control of OsGA3ox2 [170]. OsYAB1 was shown todirectly repress the expression of this gene, potentially by bindingto a GA-responsive cis-regulatory element within its promoter.The expression of OsYAB1 transcripts was found to increaserapidly in rice seedlings after GA treatment, with paclobutrazoltreatment having the opposite effect. These observations suggestthat the GA signalling-mediated changes in OsYAB1 expressionare responsible for regulating OsGA3ox2 transcript levels asopposed to a direct effect of OsYAB1 on DELLA (SLR1)-mediated transcription. Interestingly, the expression of anotherGA-regulated gene, OsGA20ox2, did not appear to be controlledby OsYAB1, supporting the existence of additional componentsresponsible for its feedback regulation.

Important recent studies have identified SCL3 (SCARECROW-LIKE 3) as a novel GA signalling component that also hasa function in regulating feedback control of GA biosynthesisin Arabidopsis [171,172]. Although SCL3 was identified as anearly GA-response gene that is positively regulated by DELLAsthrough direct binding to its promoter [9], genetic analysisdemonstrates that it acts as a positive regulator of GA-responsiveroot 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 scl3mutant [171]. Similarly, the truncated SCL3 transcript producedin this T-DNA (transferred DNA) insertion mutant (upstream ofthe T-DNA insertion) is also found to be elevated compared withwild-type plants. These observations, and the important findingthat DELLAs physically interact with SCL3, have led to a modelin which SCL3 is proposed to attenuate the transcriptional activityof DELLAs through direct interaction. Like DELLAs, SCL3is a member of the GRAS family of transcriptional regulators,but its mode of action is currently unclear. It is interestingthat SCL3 regulation of GA-dependent root growth is mediatedthrough its confined localization within the endodermis [172].This is consistent with studies demonstrating that GA signallingwithin the endodermis drives the cell elongation and cell divisionnecessary for promoting GA-dependent root growth [173,174].However, it is not entirely consistent with a role in controllingthe feedback regulation of GA biosynthesis because some ofthese genes, including GA20ox1, GA20ox2 and GA3ox1, arepredominantly expressed in other cell types within the root[175,175a]. Further work is necessary to understand themechanisms involved.

How an SCL3/DELLA complex acts to regulate the expressionof direct target genes is still unclear. ChIP (chromatinimmunoprecipitation) studies suggest that this complex bindsdirectly to regulate the expression of the SCL3 promoter [9,171],although this does not appear to be the case for the GA feedback-regulated genes GA20ox1, GA20ox2, GA20ox3 and GA3ox1. Itis conceivable that regulation of these genes occurs through analternative mechanism, for example, through the sequestrationof a transcriptional repressor. The characterization of DELLA-interacting proteins is rapidly improving our understanding ofhow they control growth regulation, and it is likely that theidentification of others will further improve this knowledge andpotentially answer the question of how they control the expressionof genes controlling GA homoeostasis.

Environmental regulation

In common with other phytohormones, GAs act as mediators ofenvironmental signals, allowing plants to respond, often rapidly,to changes in light conditions, temperature, water and nutrientstatus, and other abiotic and biotic stresses. In many cases it hasbeen shown that changes in the environment result in alteredGA content and the molecular mechanisms underlying thesemetabolic changes are beginning to be resolved, primarily inrelation to light and abiotic stress.

Depending on context, GA metabolism is sensitive to changes inlight quantity, quality or duration, which may result in increasedor decreased GA content. In early seedling development, GAsignalling suppresses photomorphogenesis [176], with exposureof dark-grown seedlings to light, i.e. de-etiolation, resultingin a rapid reduction in GA content [177]. Both phytochromeand cryptochrome light receptors are involved in this process,which results in up-regulation of GA2ox expression and, inthe case of blue light, down-regulation of GA20ox and GA3oxgenes (reviewed in [178]). Experiments with pea indicatethat the rapid decrease in GA1 content in etiolated seedlingsfollowing exposure to light, due mainly to up-regulationof PsGA2ox2 expression, required involvement of the bZIPtranscription factor LONG1, which is related to Arabidopsis HY5(ELONGATED HYPOCOTYL 5), and, like HY5, appears to beregulated at the level of protein stability by the COP1 (CON-STITUTIVELY 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, primarilythrough up-regulation of GA20ox genes (reviewed in [180]).In Arabidopsis, up-regulation of AtGA20ox2 expression inthe leaf petiole following exposure to far-red-rich LDs, in aphytochrome B-mediated process, is associated with enhancedpetiole elongation [181] and floral induction [182]. Althoughlight-induced flowering in Arabidopsis on LDs involves mainlythe CONSTANS pathway, which is independent of GAs, in theabsence of this pathway on SDs (short days), floral inductionis absolutely dependent on GAs [183]. There is evidence fromArabidopsis to suggest that, under SDs, GA4 may function as amobile signal from leaves to the shoot apex for floral induction[102], whereas in the grass Lolium temulentum, it is proposedthat the mobile signals are GA5 and GA6, produced in leavesin response to as little as one LD [184]. On account of theirunsaturation on C-2, these last GAs escape deactivation byGA2oxs 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 andelongation of the stem [126]. Compared with GA5, applied GA1

and GA4 are only weakly florigenic in L. temulentum, presumablydue to their rapid deactivation at the shoot apex [126]. In rice, aSD flowering plant, following exposure to SDs, GA2ox expressionbelow 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 enhancedexpression of StGA2ox1 adjacent to the stolon meristem prior tostolon swelling and tuber formation [185]. Transfer of potatoesto SDs also results in reduced expression of StGA3ox2 in stolons,but increased expression of this gene in shoots [186]. It is sugges-ted that in SDs less GA is transported from the shoots to the sto-lons due to enhanced conversion of the mobile GA20 into the lessmobile GA1, which accumulates and promotes shoot growth.

The red-light-induced germination of photoblastic seedsinvolves accumulation of GA through enhanced synthesis



Figure 5 Regulation of seed germination in Arabidopsis by red light andcold

The bHLH transcription factor PIL5, acting via the transcription factors DAG1 and SOM,suppresses germination by inhibiting GA biosynthesis and promoting GA deactivation, therebystabilizing DELLA proteins. Red light induces PIL5 degradation via phytochrome-catalysedphosphorylation. Stratification (cold treatment) acts to promote GA biosynthesis by preventingsuppression by the bHLH transcription factor SPT. The dark grey lines indicate feedbackregulation of GA biosynthesis and deactivation by DELLA.

and suppressed deactivation as well as a reduction in ABAcontent [187]. In Arabidopsis, this process is mediated by thebHLH transcription factor PIL5 (PIF3-LIKE 5), an inhibitorof germination that is degraded by the 26S proteasome afterbeing targeted by phosphorylation through interaction withphytochromes A and B [188]. PIL5 suppresses GA accumulation(and ABA degradation) by inhibiting expression of the GAbiosynthetic genes AtGA3ox1 and AtGA3ox2, while promotingexpression of the GA-deactivating AtGA2ox2, its action beingrelieved by the light-stimulated degradation (Figure 5). Twointermediaries in this pathway have been identified: the actionof SOM (SOMNUS), a CCCH-type zinc finger protein, has thesame effect on the GA metabolic genes as PIL5, which directlyinduces its expression [189], whereas DAG1 (DOF AFFECTINGGERMINATION 1) inhibits expression of AtGA3ox1, but has noeffect on expression of AtGA3ox2 and AtGA2ox2 [190]. DAG1,expression of which is induced indirectly by PIL5, was shown tobind to the AtGA3ox1 promoter. AtGA3ox1 is also the target ofcold induction of germination (stratification) [191], expressionof the gene being suppressed in non-stratified seeds in thedark 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, growthreduction is a common strategy that allows resources to befocused on withstanding the stress [193]. Many hormonesignalling pathways have been implicated in stress responses,including the accumulation of DELLA proteins, which restraingrowth and promote stress resistance [194], although it isunclear to what extent these responses are causally related.An emerging theme is stress-induced up-regulation of GA2oxgene expression as a means of reducing GA content andallowing DELLA accumulation. Response to abiotic stress ismediated 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,

Figure 6 Inhibition of growth in Arabidopsis by salt and cold stress throughinduction of GA deactivation

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 concentrationand greater DELLA stability. The dark grey arrows denote transcriptional promotion of the DELLAgene RGL3 by DREB/CBF-type transcription factors.

high salt-induced expression of six GA2ox genes, one of which,AtGA2ox7, was up-regulated specifically by the salt-responsiveCBF/DREB1 protein DDF1 (DWARF AND DELAYEDFLOWERING 1) [196]. DDF1 was shown to bind to a DRE-like motif on the promoter of AtGA2ox7. Short-term exposureof Arabidopsis plants to cold resulted in increased expression ofthree GA2ox genes, two of which, AtGA2ox3 and AtGA2ox6,were also up-regulated by overexpression of the cold-responsiveCBF1/DREB1b gene [197]. Cold and CBF1 overexpression alsoincreased expression of several GA biosynthetic genes, but thiseffect was abolished in a DELLA-deficient mutant, indicating thatit occurred indirectly due to feedback regulation. Although bothsalt and cold stress stimulated expression of many GA2ox genes,the tested CBF/DREB transcription factors mediated inductionof only subsets of these, indicating a high level of specificity andpresumably the involvement of other transcription factors. As wellas the accumulation of DELLA proteins resulting from reducedGA concentrations, it was shown that both salt and cold treatmentsup-regulated expression of RGL3 (RGA-LIKE PROTEIN 3),which encodes one of the five Arabidopsis DELLA proteins, theinduction being mediated by the respective CBF/DREB factors[196,197]. Thus abiotic stress allows DELLAs to accumulatethrough protein stabilization and enhanced gene expression.

Seed dormancy in Arabidopsis in response to low temperaturewas shown recently to be associated with increased expression ofAtGA2ox6, a process which is mediated by the dormancy factorDOG1 (DELAY OF GERMINATION 1) [198]. Interestingly,both DOG1 and AtGA2ox6, as well as other GA2ox genes, wereregulated by CBF1 in developing seeds, but this transcriptionfactor was not cold-induced in this context, although it appearedto have a role in seed dormancy.

The positive growth response of plants to increasing ambienttemperature was shown to be mediated by several hormonepathways, including GA signalling, with AtGA3ox1, AtGA20ox1and AtGA2ox1 identified as targets in Arabidopsis seedlings[199]. Plant growth is reduced under abnormal thermoperiodicconditions, i.e. lower temperatures during the light period than



during the dark, a condition known as negative DIF (differencebetween day temperature and night temperature) that is exploitedfor control of plant stature [200]. Stavang et al. [201] showedthat stems of pea plants grown in negative DIF contained lowerconcentrations of C19-GAs and increased expression of PsGA2ox2compared with plants grown in positive DIF and suggested thatthis 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 mostof the plant growth cycle and in response to environmentalstimuli. Recent research on GA metabolism has been directedtowards understanding regulation, which occurs primarily onthe steps catalysed by ODDs. Many molecular components thatmediate GA metabolic regulation have been identified, althoughour understanding of these processes is far from complete. Inparticular, the mechanisms underlying GA homoeostasis areproving surprisingly complex, perhaps at least in part as theresult of the promiscuous interactions of the DELLA proteins,which mediate this process. Understanding the nature of DELLA-mediated signalling and identifying the many partners of theseproteins is currently a rapidly advancing area of research. Thereis emerging evidence that DELLAs may act as a point ofconvergence for different hormone signalling pathways, therebyproviding a channel by which these pathways may influence GAmetabolism. However, it is clear that auxin signalling and, incertain circumstances also that of ABA, modify GA biosynthesisdirectly without the intervention of DELLAs [132,136]. Detailsof these mechanisms at the molecular level should become clearerin 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 GAsmay act remotely from their source is stimulating renewed interestin GA movement, which may occur over long distances withinthe vasculature, particularly the phloem, or by diffusion betweenneighbouring cells. Improved physicochemical and molecularmethods are required to probe GA biosynthesis and action atgreater tissue resolution than is currently possible.

Although GA signalling influences most aspects of plantdevelopment, the specificity of expression domains andconsequently physiological function of the ODD paralogues inGA metabolism has enabled the identification of GA-deficientmutants with desirable agricultural traits. For example, the sd1mutant of rice, lacking OsGA20ox2, and le pea, with a mutantallele of PsGA3ox1, have reduced stem height, but normal fertility.Reverse genetic approaches such as TILLING (Targeting InducedLocal Lesions in Genomes) provide an opportunity to introducesuch beneficial mutations into many more agriculturally importantspecies, although a fuller understanding of the physiological rolesof the target genes is required. The rapidly expanding number ofplant genome sequences available is simplifying the task of geneidentification, allowing suitable targets to be selected for thispurpose. Furthermore, the analysis of natural genetic variation inthese genes between varieties or ecotypes will provide importantinformation on their influence on plant phenotype. These andother developments are likely to enhance the already substantialimpact that GA-related research has had on global agriculture.

FUNDING

Rothamsted Research receives strategic support from the Biotechnology and BiologicalSciences Research Council, U.K.

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Received 9 February 2012/1 March 2012; accepted 2 March 2012Published on the Internet 26 April 2012, doi:10.1042/BJ20120245


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