Fruit Growth in ArabidopsisOccurs via DELLA-Dependent andDELLA-Independent Gibberellin ResponsesW OA

Sara Fuentes,a,1 Karin Ljung,b Karim Sorefan,a,2 Elizabeth Alvey,a,3 Nicholas P. Harberd,c and Lars Østergaarda,4

a Department of Crop Genetics, John Innes Centre, Norwich, Norfolk NR4 7UH, United KingdombDepartment of Forest Genetics and Plant Physiology, Umea Plant Science Centre, S-901 83 Umea, SwedencDepartment of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom

Fruit growth and development depend on highly coordinated hormonal activities. The phytohormone gibberellin (GA)promotes growth by inducing degradation of the growth-repressing DELLA proteins; however, the extent to which DELLAproteins contribute to GA-mediated gynoecium and fruit development remains to be clarified. Here, we provide an in-depthcharacterization of the role of DELLA proteins in Arabidopsis thaliana fruit growth. We show that DELLA proteins are keyregulators of reproductive organ size and important for ensuring optimal fertilization. We demonstrate that the seedless fruitgrowth (parthenocarpy) observed in della mutants can be directly attributed to the constitutive activation of GA signaling. Ithas been known for >75 years that another hormone, auxin, can induce formation of seedless fruits. Using mutants withcomplete lack of DELLA activity, we show here that auxin-induced parthenocarpy occurs entirely through GA signaling inArabidopsis. Finally, we uncover the existence of a DELLA-independent GA response that promotes fruit growth. Thisresponse requires GIBBERELLIN-INSENSITIVE DWARF1–mediated GA perception and a functional 26S proteasome andinvolves the basic helix-loop-helix protein SPATULA as a key component. Taken together, our results describe additionalcomplexities in GA signaling during fruit development, which may be particularly important to optimize the conditions forsuccessful reproduction.

INTRODUCTION

Phytohormones coordinate multiple aspects of plant growth anddevelopment, including fruit initiation, which is a momentous stepin the dispersal and survival strategies of flowering plants(angiosperms). Fruit initiation has traditionally been attributed tothe action of three hormones, namely, auxin, gibberellin (GA), andcytokinins (Gillaspy et al., 1993). Accordingly, application of thesehormones either alone or in combination can induce fruit growtheven in the absence of fertilization (parthenocarpy) across a widevariety of plant species (Gustafson, 1936; King, 1947; Srinivasanand Morgan, 1996; Vivian-Smith and Koltunow, 1999; Ozga et al.,2002; Ozga and Reinecke, 2003). In recent years, many of themolecular and genetic mechanisms underlying the action ofphytohormones in fruit initiation have been identified, uncoveringthe complexity of this regulatory network. Several lines of evi-dence have shown that upon fertilization, a seed-originating auxin

signal is generated (Rotino et al., 1997; Mezzetti et al., 2004;Dorcey et al., 2009). This fertilization-dependent auxin signal isproposed to upregulate GA biosynthesis, which in turn activatesGA signaling in the ovules and valves, thereby stimulating fruitgrowth (van Huizen et al., 1995; Ngo et al., 2002; Serrani et al.,2008; Dorcey et al., 2009; Ozga et al., 2009).GA metabolism highlights the complexity of the hormonal reg-

ulation of fruit development. GA biosynthesis is tightly regulatedthrough the action of two multigenic families encoding GA 20-oxidases (GA20ox) and GA 3-oxidases (GA3ox) (Chiang et al.,1995; Phillips et al., 1995; Xu et al., 1995). These enzymes catalyzeconsecutive steps of GA biosynthesis leading to bioactive GAproduction. GA metabolism also comprises GA-inactivatingpathways, and the major GA-inactivating pathway, the 2b-hydroxylation pathway, involves the activity of GA 2-oxidases(GA2ox) (Thomas et al., 1999). GA homeostasis in fruit initiationand development is determined through feedback regulation ofGA biosynthesis and catabolism. Whereas expression of mostGA20ox and GA3ox genes is downregulated in response to ele-vated bioactive GA levels or increased GA signaling, the oppositeis true for GA2ox gene expression (Hedden and Phillips, 2000; Dilland Sun, 2001; Silverstone et al., 2001; Olszewski et al., 2002;Zentella et al., 2007; Rieu et al., 2008; Achard and Genschik, 2009).Upon fertilization, expression of GA biosynthesis genes was foundto be upregulated in the ovules, whereas GA2ox expression wasdownregulated in ovules and fruit valves (Dorcey et al., 2009).In addition to GA metabolism, GA signaling is also tightly reg-

ulated during fruit development. Central to GA signaling areDELLA proteins, which are localized in the nucleus and charac-terized by a conserved DELLA motif in their N-terminal domainand a SCARECROW-like C-terminal domain (Peng et al., 1997;

1Current address: Department of Genetics, Evolution, and Environment,University College London, Gower Street, London WC1E 6 BT, UnitedKingdom.2Current address: School of Biological Sciences, University of EastAnglia, Norwich NR4 7TJ, United Kingdom.3Current address: Department of Plant Sciences, University of Cambridge,Cambridge CB2 3EA, United Kingdom.4 Address correspondence to lars.ostergaard@jic.ac.uk.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Lars Østergaard (lars.ostergaard@jic.ac.uk).W Online version contains Web-only data.OAOpen Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.112.103192

The Plant Cell, Vol. 24: 3982–3996, October 2012, www.plantcell.org ã 2012 American Society of Plant Biologists. All rights reserved.

Silverstone et al., 1998). DELLAs form part of the wider GRASfamily of regulatory proteins (Bolle, 2004) and five DELLA geneshave been identified in Arabidopsis thaliana (GA-INSENSITIVE[GAI], REPRESSOR OF GA1-3 [RGA], RGA-LIKE1 [RGL1], RGL2,and RGL3) (Peng et al., 1997; Silverstone et al., 1998; Sanchez-Fernandez et al., 1998; Dill and Sun, 2001; King et al., 2001; Leeet al., 2002; Wen and Chang, 2002). According to the relief ofrestraint model (Harberd, 2003), DELLA proteins act as growthrepressors and GA-mediated DELLA degradation is required toovercome this restraint. In agreement with their function asgrowth repressors, it has previously been reported that reductionin DELLA activity can promote parthenocarpic fruit growth (Martíet al., 2007; Dorcey et al., 2009).

At low GA levels, DELLA proteins impair the activity of basichelix-loop-helix transcription factors by interacting with their DNAbinding domain (de Lucas et al., 2008; Feng et al., 2008). Thebinding of GA to the GA receptor GIBBERELLIN-INSENSITIVEDWARF1 (GID1) promotes interaction of GID1 with the DELLAdomain of DELLA proteins (Griffiths et al., 2006; Ueguchi-Tanakaet al., 2007; Willige et al., 2007; Murase et al., 2008). The GID1-GA-DELLA complex is subsequently recognized by the SCFSLY1/GID2

E3 ubiquitin-ligase complex, which mediates ubiquitination ofDELLA proteins. This ubiquitin mark destines the DELLA proteinsfor degradation via the 26S proteasome, thereby releasing theirinhibitory interaction with transcription factors and allowing growth(McGinnis et al., 2003; Sasaki et al., 2003; Dill et al., 2004; Fu et al.,2004; de Lucas et al., 2008; Feng et al., 2008; Arnaud et al., 2010).In recent years, a proteolysis-independent mechanism of DELLAinactivation has also been described, showing that overexpressionof GID1 can rescue some of the phenotypes observed in sleepy1(sly1) mutants without affecting DELLA protein levels (Ariizumi et al.,2008). However, although GA-mediated GID1–DELLA proteininteraction is sufficient to stimulate some GA responses, 26Sproteasome–mediated DELLA degradation is still required to restoremost DELLA-dependent GA responses (Ariizumi et al., 2008). Thus,the core GA responses are still likely to rely on both GID1-mediatedGA reception and 26S proteasome–mediated degradation.

Here, we aim to obtain an in-depth understanding of GA sig-naling in the context of gynoecium and fruit development. Toachieve this, we systematically analyzed the role of DELLA pro-teins by studying fruit development in the Arabidopsis global-DELLA (gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-4) mutant, which lacks allfive DELLA proteins (hereafter referred to as the global mutant).Our results show that DELLA proteins play an important role indetermining both reproductive organ size and the size of tissuesinvolved in the promotion of fertilization. We show that the par-thenocarpic development observed in emasculated dellamutants(i.e., facultative parthenocarpy) can be directly attributed to theconstitutive activation of GA signaling, and, based on a detailedgenetic analysis, we conclude that the DELLA proteins RGA, GAI,RGL1, and RGL2 play partially redundant roles in the regulation ofpistil growth. Our analyses also reveal that auxin-induced par-thenocarpic fruit formation occurs upstream of GA signaling andis exclusively dependent on a functional GA signaling machinery.Finally, we describe and characterize a DELLA-independent GAresponse, which demonstrates that GA signaling during fruitdevelopment is more complex than previously described anduncouples a GA response from the action of DELLA proteins.

RESULTS

DELLAs Regulate Gynoecium, Style, andStigma Development

The Arabidopsis gynoecium (or pistil) is a complex structure,which upon fertilization forms the fruit. It is divided into distincttissues, such as the stigma, style, and ovary (Figure 1A). Thesetissues are made up of several different cell types (Roeder andYanofsky, 2006), and successful reproduction therefore relieson a highly coordinated developmental program to ensure effi-cient fertilization of the ovules followed by the development anddispersal of the seeds.In order to study the role of DELLA proteins throughout the

different stages of fruit development, we compared gynoecium,style, and stigma development between the wild type and thequintuple global mutant (lacking all five DELLA proteins; seeMethods for details). Several studies have shown that DELLAproteins are involved in positive regulation of GA biosynthesis,suggesting that mutants lacking DELLA proteins have low levelsof biologically active GAs (Hedden and Phillips, 2000; Silverstoneet al., 2001; Dill and Sun, 2001; Olszewski et al., 2002; Zentellaet al., 2007; Achard and Genschik, 2009). Nevertheless, to testwhether such reduced GA levels could cause growth abnormali-ties even in the absence of DELLAs, the sextuple mutant ga1global in which global is combined with the GA biosynthesismutant ga1-3 (Sun et al., 1992) was also used throughout thisstudy. In agreement with a previous study, using the quadruple-DELLA mutant lacking GAI, RGA, RGL1, and RGL2 (Cheng et al.,2004), we noticed that ga1 global mutants exhibited slightly lon-ger fruits than the wild type at stages 12 and 15 (developmentalstages defined in Smyth et al., 1990) (Figure 1B). After stage 15,the overall fruit length of both global and ga1 global was reducedcompared with the wild type (Figure 1B). Reduced fruit length isa phenotype often associated with inefficient fertilization (Vivian-Smith et al., 2001; Cox and Swain, 2006). Accordingly, we foundthat both mutants contained aborted ovules (Figure 1C), andquantification of seed set in both self- and hand-pollinated globaland ga1 global mutants showed significantly reduced seed pro-duction (Figure 1D).It has previously been suggested that absence of RGL1, RGL2,

and RGA is sufficient to restore normal seed set in the ga1-3mutant (Cheng et al., 2004). However, in this study, careful hand-pollination of ga1 global with ga1 global or wild-type Landsbergerecta (Ler) pollen did not restore normal seed set (Figure 1D).Hand-pollination of Ler pistils with ga1 global pollen did notrestore normal seed set either (Figure 1D), demonstrating that thereduced fertility in ga1 global is due to both maternally and pa-ternally derived defects. The paternally derived fertility defectsobserved in ga1 global could be caused either by abnormal pollentube growth or defects in anther and/or pollen development. In-terestingly, both global and ga1 global exhibited expanded stylelength and widened stigmatic tissue compared with the wild type(Figures 1E to 1H), and it is therefore possible that the DELLAproteins regulate growth of these tissues to allow efficient fertil-ization. Furthermore, application of GA3 to emasculated wild-typepistils mimicked the style length promotion observed in globalplants (see Supplemental Figure 1 online), confirming that style

Gibberellin Signaling in Arabidopsis Fruit Growth 3983

and stigma growth is subject to GA-mediated regulation byDELLA proteins. Based on this phenotypic analysis, we concludethat DELLA proteins are required to mediate optimal growth offruit tissues and ensure efficient fertilization.

DELLA Genes Show Distinct Expression Profiles throughoutArabidopsis Fruit Development

Previous studies in Arabidopsis have shown that individual DELLAproteins can contribute differently to distinct developmental pro-grams. For example, RGA and GAI proteins function in manyvegetative processes, such as stem elongation (Dill and Sun, 2001;King et al., 2001), whereas RGL2 is the main DELLA protein reg-ulating seed germination (Lee et al., 2002; Tyler et al., 2004).Furthermore, it was previously demonstrated that individual DEL-LA genes follow distinct developmental expression profiles inArabidopsis (Tyler et al., 2004), which can in some cases provideinformation about their relative importance in the different de-velopmental programs. Therefore, to obtain a first indication of therole played by individual DELLA proteins in fruit development, weanalyzed the expression profiles of the five Arabidopsis DELLAgenes by quantitative real-time PCR.The expression profiles of the DELLA genes followed three

different patterns during fruit development (Figures 2A to 2C).Consistent with previous data (Tyler et al., 2004), RGA expressionwas high and practically constant throughout the stages testedhere (Figure 2A). The transcript levels of GAI, RGL1, and RGL2were relatively high prior to fertilization (stage 11-12) and duringfruit elongation (stages 15 and 17a) but decreased when the finalfruit length was achieved (stage 17b) (Figure 2B). RGL3 expres-sion was high before fertilization (stages 11 and 12) but decreasedsoon after until it increased again at the final stage of fruit de-velopment (stage 17b) (Figure 2C). Although transcriptional reg-ulation is only one of the many possible mechanisms that controlDELLA protein activity (Achard and Genschik, 2009), the fact thatexpression of DELLA genes follows different profiles throughoutArabidopsis fruit development suggests that different DELLAgenes might be important at different stages of fruit growth.

Lack of DELLA Proteins Causes Facultative Parthenocarpyin Arabidopsis

The role of DELLA proteins in fruit growth repression is clearlynoticeable upon global and ga1 global emasculation. Emascu-lation of wild-type pistils 2 d before anthesis did not promotesignificant elongation, while emasculated global and ga1 globalpistils developed into parthenocarpic fruits more than twice thelength of wild-type emasculated pistils (Figure 2D).This is consistent with previous studies demonstrating that si-

lencing of a single DELLA gene in tomato (Solanum lycopersicum)and lack of four DELLA proteins in Arabidopsis result in parthe-nocarpic fruit growth (Martí et al., 2007; Dorcey et al., 2009).However, little is known about the relative importance of the in-dividual DELLA proteins in parthenocarpic fruit growth. The ex-pression patterns obtained in our study do not provide sufficientinformation to elucidate which of the DELLAs have a greatercontribution to this process. Therefore, a detailed analysis of theparthenocarpy-conferring capacity of different della mutant com-binations was performed.

Figure 1. Fertility and Fruit Development in global and ga1 global Mu-tants.

(A) Scanning electron micrograph of stage 13 Arabidopsis gynoeciumwith stigmatic tissue (sg), style (st), and ovary indicated. Bar = 400 µm.(B) Fruit development in Ler, global, and ga1 global mutant plantsthrough stages 11-12, 15, 17a, and 17b. Values significantly differentfrom those of Ler fruits are indicated by asterisks (**P < 0.01 and ***P <0.001, Student’s t test). Error bars: 95% confidence interval (CI). n $ 12.(C) Reduced number of seeds in self-pollinated global (glob) and ga1global (gg) fruits compared with Ler.(D) Reduced seed set in self-pollinated, hand-pollinated, and cross-pollinatedglobal (glob) and ga1 global (gg) mutants. Values significantly different fromthose of self-pollinated Ler are indicated by asterisks (***P < 0.001, Student’s ttest). Error bars: 95% CI, n $ 10. Red, self-pollinated; blue, hand-pollinated.(E) Scanning electron micrographs of stage 12 gynoecia from Ler, global(glob), and ga1 global (gg). Bars = 100 µm.(F) Schematic representation of style length (sl) and stigma width (sw) ofstage 12 gynoecia.(G) and (H) Quantification of pistil phenotypes. Style length (G) andstigma width (H) of stage 12 gynoecia from Ler, global (glob), and ga1global (gg). Values significantly different from those of Ler are indicatedby asterisks (***P < 0.001, Student’s t test). Error bars: 95% CI, n $ 37.

3984 The Plant Cell

Analysis of single Arabidopsis della mutants showed that onlyemasculation of rgl1 resulted in slight but significant fruit elon-gation (Figures 2E and 2F; see Supplemental Figure 2 online),while in double mutant combinations, only emasculated rgl1 rgl2pistils grew significantly longer than the wild type (Figures 2Eand 2F; see Supplemental Figure 2 online). However, an additiveeffect was observed upon emasculation of multiple della mu-tants, and emasculation of quadruple-DELLA, global, and ga1global pistils resulted in substantial parthenocarpic fruit growth(Figures 2E and 2F; see Supplemental Figure 2 online). Thus,lack of at least four DELLA proteins is required for a sizeableparthenocarpic fruit growth promotion, suggesting a certaindegree of functional redundancy. Although RGL3 expressiondiffered from that of the other four DELLA genes (Figure 2C), nosignificant difference was observed between parthenocarpicquadruple-DELLA and globalmutants (Figures 2E and 2F). Thus,GAI, RGA, RGL2, and particularly RGL1 are likely to play themain role in restricting fruit growth in the absence of fertilizationwith at most a very minor contribution from RGL3.

Tissue Structure in Parthenocarpic della Mutant Fruit WallsResembles the Structure of Pollinated and GA-TreatedWild-Type Fruits

To analyze the cellular basis underpinning the growth observed inparthenocarpic global and ga1 global fruits, we compared longi-tudinal sections of wild-type and mutant fruit walls (valves).Arabidopsis valves are composed of six cell layers: an outerexocarp layer, three layers of mesophyll cells, and two endocarplayers (endocarp a [innermost] and endocarp b). In contrast withemasculated wild-type pistils where the different tissue layerswere not clearly differentiated, tissue layers in global and ga1global parthenocarpic fruits were easily distinguishable and re-sembled the structure of pollinated or GA3-treated wild-type fruits(Figure 3A). By measuring the cell length in each individual tissuelayer of emasculated wild-type pistils and parthenocarpic globaland ga1 global fruits, we found that lack of DELLA proteins led tosignificantly longer cells across all tissue layers (Figure 3B; sta-tistical analysis in Supplemental Figure 3A online). Moreover, GA3

treatment of emasculated wild-type pistils induced cell elongationacross all tissue layers to a similar extent as pollination and as theemasculation of global and ga1 global pistils (Figure 3B), sug-gesting that GA-mediated DELLA degradation is responsible forthe parthenocarpy observed in these fruits.Induction of growth can also occur through cell division. We

therefore quantified cell numbers in the individual tissue layers ofwild-type and mutant fruits. This analysis showed that only me-sophyll and endocarp a tissue layers of parthenocarpic global andga1 global fruits had significantly higher cell numbers whencompared with emasculated wild-type pistils (Figure 3C; seeSupplemental Figure 3B online), suggesting that lack of DELLAproteins may have a greater effect on cell elongation than on celldivision.Together, these data demonstrate that GA treatment/lack of

DELLA promotes fruit growth in all layers. All layers respond andelongate in a coordinated fashion. However, different layers havespecific responses in the extent of cell division versus cell elon-gation. The exocarp elongates mainly through cell elongation,

Figure 2. Facultative Parthenocarpy in della Mutants.

(A) to (C) Expression profiles of DELLA genes through stages 11-12, 15,17a, and 17b of Arabidopsis fruit development. RGA (A), GAI, RGL1, andRGL2 (B), and RGL3 (C). Relative gene expression levels were calculatedby quantitative RT-PCR as described in Methods. Error bars indicate SD,n = 3.(D) Lack of DELLA proteins causes parthenocarpic fruit developmentin Arabidopsis flowers. Representative emasculated (E) and hand-pollinated (HP) Ler, global, and ga1 global gynoecia 7 DPA. Bar = 5 mm.(E) Representative emasculated Ler, rgl1, quadruple-DELLA (quad ),global (glob), ga1 global (gg), Columbia-0 (Col), and rgl1 rgl2 gynoecia 7DPA. Bar = 3mm.(F) Facultative parthenocarpic della mutants. Note that the gai mutationis in the tt1mutant background. Hence, for mutants with the gaimutation(quad, global, and gg), both tt1 and Ler are included as controls. The rgl1rgl2 (rgl1/2) mutant is in the Col-0 background. Asterisks indicate valuessignificantly different from control emasculated pistils (*P < 0.05 and***P < 0.001, Student’s t test). Emasculated pistils were measured 7 DPA.Error bars: 95% CI, n $ 19.

Gibberellin Signaling in Arabidopsis Fruit Growth 3985

whereas mesophyll and endocarp a layers both elongate andpromote cell division, thus increasing the number of cells.Therefore, absence of DELLA activity in the pistil is sufficient toinduce appropriate cell differentiation events similar to those ob-served upon pollination.

Auxin-Induced Fruit Elongation Requires GA Signaling

Application of the plant hormone auxin has long been knownto induce parthenocarpic fruit development in a range of plantspecies (Gustafson, 1936; Rotino et al., 1997). Several studieshave tried to unravel the basis for this effect, and it has beensuggested that auxin is produced in the ovules upon fertilizationwhich in turn induces fruit growth by promoting GA biosynthesisin other parts of the fruit (Ozga et al., 2009). The complete lack ofDELLA activity in the global and ga1 global mutants provides anexcellent system to address this hypothesis directly.Although emasculation of global and ga1 global pistils resulted in

significant parthenocarpic fruit development, these fruits wereshorter than pollinated mutant and wild-type fruits (Figure 2D);however, since pollination of mutant pistils results in growth pro-motion, they clearly have the potential to elongate. First, we testedwhether externally applied auxin could stimulate further fruit growthin these mutants. As previously reported, application of 0.1 or 1mM indole-3-acetic acid (IAA) significantly promoted elongation inemasculated wild-type pistils (Vivian-Smith and Koltunow, 1999;Figure 4A). By contrast, global and ga1 global pistils did not re-spond to IAA treatment (Figures 4B and 4C). These data suggestthat auxin-induced parthenocarpy occurs entirely through DELLA-dependent GA signaling. Second, we investigated whether theparthenocarpy observed in the della mutants is accompanied bychanges in auxin concentrations and performed direct measure-ments of the naturally occurring auxin, IAA. Emasculated pistilsfrom wild-type plants contained fivefold less IAA per mg tissue thandid the hand-pollinated fruits (Figure 4D), and this difference is likelyat least partly to reflect the high level of auxin produced by de-veloping seeds (Magnus et al., 1997). Hand-pollinated global fruitshad lower IAA levels than did hand-pollinated wild-type pistils(Figure 4D), which again can be attributed to the reduced seednumber (Figures 1C and 1D). As in the wild type, IAA content inemasculated parthenocarpic global fruits was approximately five-fold lower than in pollinated global fruits, which demonstrates thatthe facultative parthenocarpy observed in della mutants is not theresult of elevated auxin levels, but a direct consequence of DELLAprotein absence. Therefore, the data suggest that the mechanisminducing parthenocarpic fruit growth in della mutants is operatingdownstream of the ovule-derived auxin signal. This finding sup-ports the previously suggested hypothesis that auxin is requiredto induce GA biosynthesis (Dorcey et al., 2009; Ozga et al.,2009), which in turn promotes DELLA degradation to relievegrowth restraint. Interestingly, the parthenocarpic global fruitsFigure 3. Tissue Analysis of Ler, global Mutant, and ga1 global Mutant

Fruits.

(A) Longitudinal sections of Ler, global (glob), and ga1 global (gg) fruits 7DPA. C, emasculated and control-treated; +GA, emasculated and 10 mMGA3-treated; P, self-pollinated. Bar = 0.2 mm.(B) Comparison of cell length parallel to the fruit elongation axis in Ler,global (glob), and ga1 global (gg) exocarp (x), mesocarp (m), and endo-carp a (a) tissue layers.

(C) Comparison of cell number normal to the fruit elongation axis in Ler,global (glob), and ga1 global (gg) exocarp (x), mesocarp (m), and endo-carp a (a) tissue layers. Error bars: 95% CI, n$ 4. Statistical analysis is inSupplemental Figure 3 online.

3986 The Plant Cell

contained significantly less IAA than did emasculated wild-typepistils (Figure 4D), which could suggest the presence of a feed-back loop in which DELLA proteins induce auxin production.

Arabidopsis Fruit Growth Is Partially Determined bya DELLA-Independent GA Response

To investigate whether elongation could be further promoted inemasculated global and ga1 global pistils, a series of hormonaltreatments were performed. In comparison to tissues such asroots and leaves, relatively high hormone concentrations arerequired to observe effects in pistils and fruits (Srinivasan andMorgan, 1996; Vivian-Smith and Koltunow, 1999; Dorcey et al.,2009), which is most likely due to a reduced absorption capacitythrough their waxy surface. To determine the effect of added hor-mone solutions under our plant growth conditions, dose–responseexperiments were performed based on previous studies (Vivian-Smith and Koltunow, 1999).

Application of GA3 promoted parthenocarpic fruit developmentin wild-type Arabidopsis plants and to a greater extent thanIAA application (Figure 5A) in agreement with previous findings(Vivian-Smith and Koltunow, 1999). Unexpectedly, significant fruitelongation was also observed in global and ga1 global upon GA3

treatment in a dose-dependent manner (Figures 5B and 5C), re-vealing the existence of a DELLA-independent GA response.GA3 is not a naturally occurring GA in Arabidopsis and, in order

to rule out that the growth promotion observed in global and ga1global pistils could be due to the artificial nature of this com-pound, pistil growth experiments were repeated with the bioactiveGA, GA4 (Ross et al., 2008). Application of GA4 to emasculatedglobal and ga1 global pistils also promoted elongation (Figure 5E),confirming the existence of a DELLA-independent GA responseduring fruit development. Lower concentrations of GA4 than GA3

were required to promote global and ga1 global pistil elongation(Figures 5B, 5C, 5E, and 5F), which is probably due to GA4 beinga naturally occurring GA in Arabidopsis while GA3 is not. Never-theless, GA3 is the GA that has traditionally been used in mostArabidopsis studies (Vivian-Smith and Koltunow, 1999); thus, inorder to allow comparisons with previous studies, GA3 was alsoemployed as the main GA in this study.To gain further insight into how the newly discovered DELLA-

independent GA response might be regulated, we tested the re-sponse of gai Dominant (gai-1) mutant pistils to GA3 application.The gai-1 mutant contains a mutated form of the DELLA proteinGAI that is resistant to GA-mediated protein degradation (Penget al., 1997). Hence, the gai-1 mutant does not respond to GAI-dependent GA signaling. In contrast with what has previouslybeen reported (Vivian-Smith and Koltunow, 1999), application ofGA3 to gai-1 pistils resulted in significant growth promotion underour experimental conditions (Figure 5D). Although contributions togrowth may occur due to degradation of other DELLA proteins inthe gai-1 mutant pistils, this result further supports the existenceof a DELLA-independent GA response in fruit development asobserved in global and ga1 global mutants.

The DELLA-Independent GA Response Requires Both theGID Receptor and the 26S Proteasome and IsSPT Dependent

Previous studies have shown that GID1 receptor-mediated GAperception and 26S proteasome–mediated DELLA degradation arenecessary for most GA responses (Fu et al., 2002; Ueguchi-Tanakaet al., 2005; de Lucas et al., 2008; Feng et al., 2008; Gao et al.,2011; Sun, 2011). Thus, whether the newly discovered DELLA-independent GA response also relies on GID1-mediated GA re-ception and 26S proteasome–mediated degradation was tested.Three GID1 GA receptors have been identified in Arabidopsis

(Griffiths et al., 2006; Nakajima et al., 2006). Genetic studieshave shown that the gid1a-1 gid1b-1 gid1c-1 (gid1a/1b/1c) triplemutant exhibits severe GA-related developmental defects, in-cluding delayed flowering time (Griffiths et al., 2006), and underour growth conditions, gid1a/1b/1c triple mutant plants onlyflowered after 2 months of vegetative development. Measure-ments in vegetative tissue demonstrated that the gid1a/1b/1cmutant accumulates high levels of GA (Griffiths et al., 2006). Tofacilitate a potential response above a high background, only the

Figure 4. Auxin-Induced Fruit Growth Is Dependent on GA Signaling.

(A) to (C) Percentage growth change in Ler (A), global (B), and ga1 global(C) in response to increasing concentrations of IAA (0.1 and 1 mM).Pistils were measured 7 DPA. Buffer solution was used as the controlsolution (0 mM). P, self-pollinated. Values significantly different fromcontrol pistils in each of the panels are indicated by asterisks (***P <0.001, Student’s t test). Error bars: 95% CI, n $ 20.(D) IAA levels measured in emasculated (white) and hand-pollinated (gray)Ler, global mutant, and ga1 global mutant fruits 7 DPA. Asterisks indicatevalues significantly different from Ler emasculated pistils (**P < 0.01 and***P < 0.001, Student’s t test), and white circles indicate values significantlydifferent from pollinated Ler fruits (oooP < 0.001, Student’s t test). Error bars:95% CI, n = 3.

Gibberellin Signaling in Arabidopsis Fruit Growth 3987

highest GA3 concentration was used to determine whether thenewly discovered DELLA-independent GA response relies onGID1-mediated GA reception. Application of 10 mM GA3 togid1a/1b/1c triple mutant pistils did not promote elongation(Figure 6A), suggesting that the DELLA-independent GA re-sponse requires GID1-mediated GA reception.In order to determine whether the 26S proteasome is also

involved in the DELLA-independent GA response, treatmentswith MG132 (a 26S proteasome-specific inhibitor) and GA3 werecombined (Figure 6B; see Supplemental Figure 4 online). Thegrowth promotion response observed upon 10 mM GA3 appli-cation was completely blocked by pretreatment with 100 µMMG132 (Figure 6B; see Supplemental Figure 4 online), demon-strating that the 26S proteasome is a necessary component ofthe DELLA-independent GA response pathway.The SLY1 gene in Arabidopsis encodes an F-box protein of the

SCFSLY1 complex. Interaction of SLY1 with DELLA proteins pro-motes their ubiquitination and subsequent degradation in the 26Sproteasome (McGinnis et al., 2003; Dill et al., 2004; Feng et al.,2008). In order to determine whether SLY1 is part of the DELLA-independent GA pathway, emasculated sly1-10 pistils weretreated with GA (see Supplemental Figure 5 online). A significantgrowth promotion was observed in GA-treated sly1-10 pistils,which suggests that SLY1 is not required for the DELLA-independent GA response (see Supplemental Figure 5 on-line). This may not be entirely unexpected as the interactionbetween F-box proteins and their target proteins conferssubstrate specificity to the SCF complex (del Pozo and Estelle,2000; Gagne et al., 2002). Thus, our results support thatSLY1 F-box protein is specifically involved in DELLA proteindegradation.In addition to the GID1 receptors and the 26S proteasome,

more recently, the basic helix-loop-helix transcription factor SPThas also been shown to be involved in GA responses. SPT notonly represses GA biosynthesis in dormant seeds (Penfieldet al., 2005) but also interacts with DELLA proteins in yeast two-hybrid assays (Gallego-Bartolomé et al., 2010; Josse et al.,2011). SPT was originally characterized as a major player in styledevelopment in the gynoecium (Alvarez and Smyth, 1999;Heisler et al., 2001). More recent studies have revealed that SPTalso functions redundantly with other factors to promote fruitopening upon maturity in a process that requires GA signaling(Arnaud et al., 2010; Girin et al., 2011; Groszmann et al., 2011). Itis therefore plausible that SPT function contributes to the newlydiscovered DELLA-independent GA response.To test the role of SPT, we obtained the quadruple-DELLA spt-2

quintuple mutant (Josse et al., 2011). The spt-2 allele results from anamino acid substitution in the putative DNA binding domain of SPT(Heisler et al., 2001; Penfield et al., 2005) and the mutant phenotypeis fully complemented by the wild-type SPT gene (Heisler et al.,2001). Here, we found that in contrast with quad-DELLA, global, andga1 global mutants, GA3 treatment of emasculated quad-DELLAspt-2 mutants did not promote fruit elongation (Figures 6C and 6D).These data therefore suggest that SPT is required for the DELLA-independent GA response. Moreover, the lack of GA-inducedgrowth in the quad-DELLA spt-2 mutant compared with the quad-DELLA mutant also provides genetic evidence for complete loss ofDELLA activity in the quad-DELLA mutant during fruit growth.

Figure 5. GA3 and GA4 Promote Fruit Elongation in Ler, global, ga1global, and gai-1.

(A) to (D) Percentage growth increase in emasculated Ler (A), global (B),ga1 global (C), and gai-1 (D) plants in response to increasing concen-trations of GA3 (0.1, 1, and 10 mM).(E) and (F) Percentage growth increase in emasculated global (E) andga1 global (F) in response to GA4 (0.01 and 0.1 mM).Pistils were measured 7 DPA. Buffer solution was used as the controlsolution (0 mM). P, self-pollinated. Values significantly different fromcontrol pistils in each of the panels are indicated by asterisks (*P <0.05, **P < 0.01, and ***P < 0.001, Student’s t test). Error bars: 95% CI,n $ 16.

3988 The Plant Cell

Comparison of emasculated spt-3, spt-11, and spt-12 mutantpistils showed that these pistils were significantly longer than wild-type emasculated pistils (Figure 6E) and, hence, partially parthe-nocarpic. Based on these data and in agreement with other studies(Ichihashi et al., 2010; Sidaway-Lee et al., 2010; Josse et al., 2011),

we hypothesize that in the absence of GA, SPT acts as fruit growthrepressor. Upon GA treatment, all spt loss-of-function allelesshowed decreased responsiveness when compared with wild-typepistils (Figure 6E). These data support that a functional SPT isrequired for the DELLA-independent GA response.Since the DELLA-dependent and DELLA-independent GA

pathways share key mechanisms such as involvement of GID1receptors and the 26S proteasome, we wondered whether SPT,like DELLA proteins, is degraded in response to GA. This possi-bility was investigated by immunoblotting analysis using a trans-genic line in which SPT is fused to the hemagglutinin (HA) epitopetag and expression is driven by the 35S promoter (Sidaway-Lee et al., 2010). Immunoblotting with an HA antibody revealedno SPT-HA protein degradation in fruits upon GA3 treatment(Figure 7A).The DELLA domain of DELLA proteins is essential not only for

GA-induced degradation, but also for interaction with the GID1receptor (Dill and Sun, 2001; Willige et al., 2007). Although SPTprotein lacks a DELLA-like domain, the possible interaction be-tween SPT and GID1A receptor was assayed using yeast two-hybrid analysis (Figure 7B). GAI and RGA proteins were used aspositive controls for GA-dependent interaction with GID1A (Figure7B; Willige et al., 2007). However, in the case of SPT, no in-teraction with GID1A could be detected (Figure 7B). In the ab-sence of GA, DELLA proteins did not interact with GID1 inagreement with previous results, and this was also the case forSPT (Willige et al., 2007; data not shown). Taken together, the lackof SPT–GID1A interaction and the lack of GA-mediated SPTdegradation demonstrate that the mode of action of SPT in GAsignaling is different from that of the DELLA proteins. These dataalso suggest that additional molecular components are involvedin the DELLA-independent GA signaling pathway.

DISCUSSION

It has previously been shown that DELLA proteins are importantregulators of floral organ growth and patterning (Cheng et al.,2004; Tyler et al., 2004; Dorcey et al., 2009; Arnaud et al., 2010). Inthis article, we further characterize the relative importance of in-dividual DELLA proteins in fruit development. We demonstratethat, although DELLA proteins act as major growth repressors,a DELLA-independent GA response during Arabidopsis fruit de-velopment also exists. This newly discovered DELLA-independentGA pathway shares several characteristics with the DELLA-dependent pathway. However, we show that in this new pathway,SPT is a key growth-repressing factor and is regulated differentlyfrom DELLA proteins during GA signaling.

DELLA Proteins Repress Gynoecium, Style, and StigmaGrowth in Arabidopsis

To investigate the role of DELLA proteins during gynoecium andfruit development in Arabidopsis, global and ga1 global mutantslacking all five DELLA proteins were analyzed throughout thedifferent stages of fruit development. We included both of thesemutants in our studies in case the results we obtained in theglobal mutant were due to abnormal GA levels. However, similarresults were obtained with these two mutants throughout.

Figure 6. Function of GID1 Receptors, 26S Proteasome, and SPT in theDELLA-Independent GA Response.

(A) Pistil length of gid1a/1b/1c triple mutant in response to 10 mM GA3.Pistils were measured 7 DPA. Buffer solution was used as the controlsolution. Error bars: 95% CI, n $ 12.(B) Blocking of the DELLA-independent response in global mutant pistilsin response to the 26S proteasome inhibitory drug, MG132 (MG). Pistilswere measured 7 DPA. Buffer solution was used as the control solution(C) and MG132 was diluted in 0.01% DMSO (D).(C) quad-DELLA spt-2 (quad spt-2) mutant pistils do not respond to in-creasing concentrations of GA3.(D) Pistil length of emasculated quad-DELLA spt-2 mutants comparedwith quad-DELLA control and 10 mM GA3 treated (**P < 0.01, Student’s ttest). Error bars: 95% CI, n $ 32.(E) Pistil length of emasculated and GA3-treated pistils of spt-2, spt-3,spt-11, and spt-12 mutants compared with Ler and Col-0 plants (**P <0.01 and ***P < 0.001, Student’s t test). Error bars: 95% CI, n $ 18.

Gibberellin Signaling in Arabidopsis Fruit Growth 3989

Detailed characterization of stage 12 gynoecia demonstratedthat lack of DELLA proteins results in enlarged style and stigmatissue development (Figure 1). Previous studies have shown thatsilencing of DELLA activity in tomato also promotes style growth(Martí et al., 2007), and the results described here for Arab-idopsis demonstrate that this effect is not limited to fleshy fruitdevelopment. Furthermore, style elongation could be phe-nocopied in wild-type Arabidopsis pistils upon GA treatment(see Supplemental Figure 1 online), supporting the hypothesis thatstyle development is under the control of DELLA-dependent GAsignaling.

The importance of DELLA-dependent GA signaling in gy-noecium and fruit development is further supported by the ob-servation that emasculation of global and ga1 global pistilspromotes parthenocarpic fruit development (Figure 2). It haspreviously been reported that emasculation of a quadruple-DELLA Arabidopsismutant also results in fertilization-independentfruit growth (Dorcey et al., 2009). However, despite the numerousstudies underlining the functional specialization of individualDELLA proteins (Peng et al., 1997; Silverstone et al., 1998; Dilland Sun, 2001; King et al., 2001; Lee et al., 2002; Tyler et al.,2004; Cao et al., 2005), the role that individual DELLA genesmay play during fruit development remains poorly understood.

Transcript level analysis showed that their expression profilesfollow three different patterns during fruit development (Figures2A to 2C), suggesting that different DELLA genes may be im-portant at different stages of fruit growth. For example, RGL3expression decreases after fertilization but increases when themaximum fruit length is achieved at stage 17b (Figure 2C). Al-though the rgl3 single mutant has no detectable defect in re-stricting fruit growth, it is possible that RGL3 functions redundantlywith other factors at this stage to determine final fruit length.DELLA activity is subjected to regulation at many different levels(Achard and Genschik, 2009); however, this expression analysis isin accordance with previous studies, which emphasize the rele-vance of the transcriptional regulation of DELLA genes (Tyler et al.,2004; Oh et al., 2007).

A detailed analysis of the parthenocarpic capacity of differentdella mutant combinations was also performed in order to un-derstand the relative importance of individual DELLA proteins inthe control of fruit initiation (Figure 2; see Supplemental Figure 2online). After emasculation of single mutant flowers, only rgl1exhibited significant fruit growth promotion and additional lackof DELLA proteins is required to enhance this effect (Figure 2;see Supplemental Figure 2 online). However, no difference wasobserved between parthenocarpic quadruple-DELLA and globalmutants (Figure 2), suggesting at most a very minor contributionfrom RGL3 in fruit growth repression. Thus, GAI, RGA, RGL2,and particularly RGL1 are likely to play the main role in theregulation of fruit development.At the tissue level, initial analysis showed that lack of DELLA

proteins in parthenocarpic global and ga1 global fruits resulted insignificant cell elongation across all tissue layers (Figure 3).However, only inner tissue layers of parthenocarpic global andga1 global fruits showed a greater cell number (Figure 3C). Al-though GA action has traditionally been associated with celllength promotion (Srivastava et al., 1975; Davidonis, 1990; Inadaand Shimmen, 2000; Cheng et al., 2004; Ubeda-Tomás et al.,2008), more recently it has also been shown that DELLA growth-repressing action involves the reduction of both cell proliferationand expansion rates in Arabidopsis roots (Achard et al., 2009;Ubeda-Tomás et al., 2009). Based on our observations alone, weare not able to conclude whether lack of DELLA proteins affectscell elongation and division to a similar extent in the differenttissue layers or whether there is a primary GA-responsive tissueduring fruit development. The use of cell layer–specific attenua-tion of the GA response could undoubtedly provide the means toanswer this question as shown by Ubeda-Tomás et al. (2008),who proved that DELLA-dependent GA signaling in the endo-dermis is the rate-limiting factor during root elongation.

Auxin Functions Upstream of GA and DELLAs to InduceFruit Elongation

It has been known for decades that parthenocarpic fruit de-velopment can be induced by manipulating hormone levels infruits, either through direct application or by genetic engineering(Gustafson, 1936; Wittwer et al., 1957; Gillaspy et al., 1993;Vivian-Smith and Koltunow, 1999). One explanation for this effectcould be that the added hormone compensates for the hormonenormally produced by the seeds. Indeed, elevated levels of GAand auxin have been measured in fruits from plants that exhibitparthenocarpy in nature (Talon et al., 1990). Moreover, ectopicbiosynthesis of auxin in seeds through genetic engineering hasproven a powerful method to obtain seedless fruits in a numberof plant species (Rotino et al., 1997; Ficcadenti et al., 1999;Mezzetti et al., 2004).The direct measurement of IAA in wild-type, global, and ga1

global fruits reported here shows no elevation of auxin levels inparthenocarpic global and ga1 global fruits. This suggests thatthe mechanism inducing parthenocarpic fruit growth in dellamutants functions downstream of the ovule-derived auxin sig-nal. It has previously been proposed that auxin distribution fromthe ovule upon fertilization is required to induce GA biosynthesisin the pericarp (Dorcey et al., 2009; Ozga et al., 2009), which in

Figure 7. SPT Is Resistant to GA-Mediated Degradation and Does NotDirectly Interact with the GID1A GA Receptor

(A) STP-HA is stable upon GA3 treatment in Arabidopsis fruits. Immu-noblot showing the stability of SPT-HA upon GA3 treatment in stage 12/15 fruits. Coomassie blue staining of protein gel is shown below (ar-rowheads: position of SPT-HA at 42 kD).(B) Yeast two-hybrid interaction using GAI, RGA, and SPT as baits andGID1A as prey. Yeast cells were plated on selective medium (left panel)and control medium (right) in both cases in the presence of 100 µM GA3.Ø indicates empty vector used as negative control.

3990 The Plant Cell

turn would promote DELLA degradation to relieve growth re-straint. The data obtained here support this hypothesis.

Since we found that ectopic addition of auxin did not furtherinduce growth in the global and ga1 globalmutant backgrounds,it is unlikely that a parallel auxin-promoted route to partheno-carpy exists in Arabidopsis. We therefore propose that Arabi-dopsis fruit development induced by auxin occurs solely throughactivation of GA signaling.

The lower levels of IAA recorded in parthenocarpic globalfruits compared with emasculated wild-type pistils suggests thatDELLAs may be involved in a positive feedback loop to promoteauxin biosynthesis. Such a mechanism is plausible, as DELLAsare also known to induce expression of GA biosynthesis genes(Dill and Sun, 2001; Olszewski et al., 2002).

Evidence for a DELLA-Independent GA Response duringFruit Development

Although emasculation of global and ga1 global pistils results infruit growth promotion, the parthenocarpic fruits do not grow tothe same length as seed-bearing siliques (Figures 2D to 2F). Thisobservation suggests that pathways other than DELLA-dependentGA signaling may be involved in the regulation of Arabidopsis fruitdevelopment. The generation of a complete della knockout mu-tant, such as global and ga1 global, allows the identification ofDELLA-independent responses. Indeed, application of GA3 orGA4 to emasculated global and ga1 global pistils resulted in sig-nificant growth promotion, revealing the existence of a DELLA-independent GA response.

Application of GA3 to dominant gai-1 mutant pistils also pro-motes elongation, supporting the DELLA independence of thenewly discovered response. Although we cannot discard thepossibility that the growth promotion observed in gai-1 pistilscould be partially mediated by degradation of the remainingfunctional DELLA proteins, this is unlikely since a GA responsein gai-1 has not previously been reported.

The DELLA-Independent GA Response Shares SomeCommon Components with the DELLA-Dependent Pathway

Degradation of proteins through ubiquitination and relocation tothe 26S proteasome is a common feature in most hormone-signaling pathways (Santner and Estelle, 2009). This mechanismrequires interaction between specific protein domains oftenmediated by direct interactions of the hormone with its receptor(s).In agreement with this, our analyses demonstrate that the DELLA-independent GA pathway, similarly to the DELLA-dependentpathway, requires both the 26S proteasome and most likely theGID1 receptors to exert its effect.It is interesting to notice that application of the 26S protea-

some inhibitor MG132 alone to emasculated global and ga1global pistils also resulted in growth reduction when comparedwith control-treated pistils (Figure 6B). It has previously beenshown that unpollinated gynoecia continue to grow after emas-culation and increase in length up to one-half of their originalanthesis length (Vivian-Smith and Koltunow, 1999; Carbonell-Bejerano et al., 2010). Thus, our results suggest that blockingthe 26S proteasome is likely to affect both the postanthesisgrowth of unpollinated pistils and the DELLA-independent GAresponse.Overall, we can conclude that GID1 receptors and the 26S

proteasome are components shared between the DELLA-dependent and DELLA-independent GA pathways. However, wefound that the two pathways most likely branch out at the level ofSCF complex specificity, since the DELLA-independent pathwaydid not rely on functional SLY1 F-box protein.

SPT as a Growth Repressor in the DELLA-IndependentGA Pathway

Studies of SPT in fruit development have revealed its role ingynoecium patterning and valve margin formation (Alvarez andSmyth, 1999, 2002; Heisler et al., 2001; Girin et al., 2011).

Figure 8. Model for GA Signaling during Fruit Development.

DELLA and SPT (together with an unidentified factor X) act as fruit growth repressors prior to fertilization. Upon fertilization, an ovule-derived auxinsignal induces GA biosynthesis, which promotes two pathways: (1) The DELLA-GA-GID1 complex is formed, resulting in fruit growth promotion through26S proteasome–dependent DELLA degradation; (2) GA perception enables the fine-tuning of fruit growth through a DELLA-independent pathway thatrelieves SPT repression possibly by dissociation from X, which is then targeted for degradation in the 26S proteasome.

Gibberellin Signaling in Arabidopsis Fruit Growth 3991

Several lines of evidence show that SPT is also involved in GAsignaling: SPT represses GA biosynthesis during etiolated hypo-cotyl growth (Penfield et al., 2005), SPT and DELLA proteins caninteract in yeast two-hybrid experiments (Gallego-Bartoloméet al., 2010), and SPT and DELLAs have dual action in the regu-lation of cotyledon growth (Josse et al., 2011). In this study weshow that in contrast with quad-DELLA and global mutants, ap-plication of GA3 to emasculated quad-DELLA spt-2 pistils doesnot result in growth promotion, confirming that SPT also acts asa growth repressor in the DELLA-independent GA pathway. Thisis further supported by our data that spt loss-of-function mutantsare partially parthenocarpic and fail to elongate to the same extentas emasculated wild-type pistils upon GA treatment.

The role of SPT in fruit growth repression is in accordancewith recent reports showing that SPT functions as a repressor ofleaf and cotyledon growth (Ichihashi et al., 2010; Sidaway-Leeet al., 2010; Josse et al., 2011). Based on its growth-repressingcapacity, we hypothesized that the role of SPT in the DELLA-independent GA pathway may be comparable to that of DELLAsin the DELLA-dependent GA pathway. However, in contrast withDELLA proteins, no GA-mediated SPT degradation was ob-served in pistils. Furthermore, GA3 did not promote GID1–SPTinteraction in yeast, suggesting that the DELLA-independentsignaling pathway is likely to rely on additional uncharacterizedmolecular players.

GA Signaling during Fruit Development

The data presented here show that DELLA proteins are vital forappropriate gynoecium and fruit development in Arabidopsisand that fruit growth requires DELLA degradation. This is inaccordance with the relief of restraint model, where activation ofGA responses requires GA-mediated DELLA protein degrada-tion by the 26S proteasome (Figure 8; Harberd, 2003). Further-more, our results reveal that auxin-mediated parthenocarpy inArabidopsis occurs entirely through stimulation of GA signaling.

In addition, we identified a GA signaling pathway that is in-dependent of DELLA inactivation but that still requires the GID1receptors and a functional 26S proteasome. We have alsoshown that SPT is a key factor in this pathway. However, sinceSPT itself is not degraded upon GA treatment, we speculate thatone or more other factors are involved. As presented in themodel in Figure 8, we hypothesize that SPT interacts with anunidentified factor X in the absence of GA to repress growth ofthe gynoecium. In the presence of GA, this interaction is dis-rupted and X is degraded in the 26S proteasome similar toDELLAs, allowing the repression to be relieved.

Our results clearly identify the classical DELLA-dependent GApathway as having the dominant effect on Arabidopsis fruit elon-gation. However, in analogy with the 26S proteasome-independentmechanism of DELLA inactivation (Ariizumi et al., 2008), theDELLA-independent pathway identified here provides addi-tional opportunities for fine-tuning fruit growth. Fruit developmentrelies on careful coordination of growth between distinct tissuetypes to allow protection of the fertilized seeds and their timelydispersal. Additional layers of regulatory complexity in this organmay therefore be particularly important to optimize the conditionsfor successful reproduction.

METHODS

Plant Material and Growth Conditions

The global-DELLA mutant (lacking all five DELLA genes) was obtained asdescribed by Koini et al. (2009). The ga1-3 global-DELLA mutant wasisolated from a cross between a ga1-3 quadruple-DELLA mutant (Chenget al., 2004) and a gai rga rgl1+/2rgl2 rgl3+/2 mutant plant. Seeds from bothmutants were kindly provided by Liz Alvey. Seeds from quadruple-DELLAspt-2mutant (Josse et al., 2011) and 35S:SPT-HA (Sidaway-Lee et al., 2010)were kindly provided byStevePenfield, andwe received thegid1a-1 gid1b-1gid1c-1 seeds (Griffiths et al., 2006) as a kind gift from Stephen Thomas.

The global-DELLAmutant consists of a combination of the gai-t6, rga-t2, rgl1-1, rgl2-1, and rgl3-4 alleles (Koini et al., 2009). Isolation andcharacterization of the first four alleles are described by Peng et al. (1997,2002), Lee et al. (2002), and Cheng et al. (2004).

In the rgl3-4 allele, the T-DNA insertion is located 1203 nucleotidesdownstream from the ATG start codon of the RGL3 gene, reducing thelength of the predicted translational product by 121 residues. In a tran-scriptional analysis wewere unable to detect a full-length transcript in rgl3-4RNA isolated from floral tissue, whereas a reduced level of a truncated rgl3-4 was detected (see Supplemental Figure 6 online). This product is ter-minated in the middle of the conserved PFYRE domain and the SAWdomain is missing completely. Based on conservation within the GRASprotein family, this truncated rgl3-4 transcript is unlikely to be translated intoa functional DELLA protein (Bolle, 2004). Furthermore, mutant alleles of thebarley DELLA gene SLENDER1 that encode proteins lacking the last fewamino acids of the full-length protein confer the slender (loss-of-function)phenotype (Chandler et al., 2002). As there is high level of sequencesimilarity and functional homology between DELLA proteins across an-giosperms, it is reasonable to assume that Arabidopsis thaliana DELLAproteins (including RGL3) also require a full-length protein to function.

Plants were grown in a controlled environment room at 20°C under 16-h-light/8-h-dark photoperiod prior to which seeds were stratified at 4°C indarkness for 4 d.

For all emasculation experiments, the first three stage 12 flowers wereemasculated. Flowers at this stage were also used for the style and stigmameasurements in Figures 1G and 1H. Hand-pollinations were performedafter emasculations throughout.

Pistil Emasculation and Hormone Treatments

Pistil emasculations and hormone treatments were performed as describedby Vivian-Smith and Koltunow (1999), and dose–response experimentswere performed to further determine the optimal hormone concentration. A1-mL droplet containing 0.1, 1, or 10 mM of GA3 or IAA with 0.01% (v/v)Triton X-100 buffered to pH 7.0 was used to coat emasculated pistils atstage 13 uniformly. Triton X-100 buffer solution (0.01% [v/v]) was used asthe control solution. During 26S proteasome inhibition experiments, a 1-mLdroplet containing 0.01% DMSO or 100 µM MG132 was applied and let todry prior to control or GA3 treatment. At least five individual plantswere usedin each of the hormone treatment experiments.

Final pistil length following pollination, hormone, or control treatment wasmeasured 7 d postanthesis (DPA) once pistils had fully elongated. Pistilswere examined under light microscopy using an MZ 16 stereomicroscope(Leica), and the images were captured with a DFC 280 digital camera (Leica).Measurements were performed on captured images using ImageJ software.

Quantitative Real-Time PCR Analysis

Total RNA was extracted from homogenized samples of stage 11-12, 15,17a, and 17b flowers and fruits using the RNeasy Plant Mini Kit (Qiagen)according tomanufacturer’s protocol. Threemicrograms of total RNAwasused to synthesize first-strand cDNA, using M-MLV reverse transcriptase

3992 The Plant Cell

(Invitrogen) and oligo(dT) (18) primers. Gene expression was determined byquantitative real-timePCR (Chromo4 real-timePCRdetector; Bio-Rad) usingthe SYBRGreen JumpStart Taq ReadyMix PCRMasterMix (Sigma-Aldrich).Expression levels of DELLA genes were calculated relative to the averageexpression level of TUBULIN8, UBIQUITIN-CONJUGATING ENZYME9, andUBIQUITIN-CONJUGATING ENZYME10 genes. The gene-specific primersequences used in this study are shown in Supplemental Table 1 online.

Endogenous IAA Quantification

IAA quantification was performed as described by Sorefan et al. (2009).For each measurement, three independent pooled samples (10 to 20 mgfresh weight) of emasculated and self-pollinated Ler and global mutantgynoecia 7 DPA were collected.

Scanning Electron Microscopy

Fruits were fixed for ;4 h at 25°C in FAA (50% ethanol, 5% glacial aceticacid, and 3.7% formaldehyde). After critical point drying, tissue wascoated with gold and examined in a Philips XL30 FEG microscope usingan acceleration voltage of 3 kV.

Histological Analysis

Pistils treated with the control solution, 10 mM GA3, or self-pollinatedpistils were collected 7 DPA for histological analysis. Tissue fixation wasperformed by vacuum infiltration with FAA solution (3.7% formaldehyde,5% acetic acid, and 50% ethanol) followed by a series of 30-min ethanolwashes (50, 60, 70, 80, and 90%). Tissue was stored overnight in 95%ethanol at 4°C. A second round of 30-min washes was performed thefollowing day (33 100% ethanol, 75% ethanol/25% Histoclear, 50%ethanol/50% Histoclear, 25% ethanol/75% Histoclear, and 33 100%Histoclear). Tissue was stored overnight in 50% Histoclear/50% Para-plast at 60°C and subsequently embedded through six changes ofParaplast at 60°C. An RM 2255 rotary microtome (Leica) was used tomake 8-mm thin longitudinal carpel sections.

To visualize the different tissue layers in the longitudinal carpel sections,tissue staining with Alcian blue 8Gx (staining unlignified tissue blue) andSafranin-O (staining lignified tissue red) was performed. After deparaffini-zation, sections were treated with Alcian Blue 8Gx/Safranin-O solution(0.05% Alcian Blue 8Gx and 0.01% Safranin-O in 0.1 M acetate buffer, pH5.0) for 20 min. Longitudinal carpel sections were examined under lightmicroscopy using a MZ16 stereomicroscope (Leica), and the images werecaptured with a DFC 280 digital camera (Leica). Cell length parallel to theplane of silique elongation was measured on captured images using ImageJ software (10 to 27 cells per tissue layer per section each from n $ 4sections). The middle part of the fruit was chosen, and measurements werealways done in the same area. These data were used to calculate the totalnumber of cells normal to the pistil elongation axis by dividing the meansilique length by the mean cell length normal to the plane of elongation.

Immunoblotting

Approximately 300 mg of plant tissue from flowers at stages 12 to 15 wascollected and grinded. One milliliter of protein extraction buffer (50 mMTris HCl, pH 7.5, 400 mM NaCl, and 1 mM EDTA) with 10 mL Sigma-Aldrich complete protease inhibitor cocktail and 0.5 mL of 200 µMMG132wasmixed thoroughly with the grinded tissue. The debris was removed bycentrifugation at 13,000g at 4°C for 10 min. The supernatant wastransferred to a fresh Eppendorf tube and stored at 280°C.

After protein extraction and quantification by Bradford assay (Bio-Rad), sample running buffer and reducing agent were added to samplesaccording to the RunBlue protocol (http://www.expedeon.com/TECHNICALRESOURCES/PRODUCTMANUALS/tabid/125/Default.aspx),and this mixture was heated for 10 min at 70°C. Protein extracts were

separated by SDS-PAGE (12%) and transferred to a nitrocellulosemembrane. A rabbit anti-HA polyclonal Chip-grade antibody (Abcam) wasapplied, and a goat anti-rabbit IgG antibody (InmunoPure Antibody;Thermo) was used as a secondary antibody to enable immunoreactivepolypeptide visualization after film development with Supersignal WestPico Chemiluminescent substrate (Thermo).

Yeast Two-Hybrid Assays

For the yeast two-hybrid assays, the GID1A-BD vector (Willige et al., 2007)used as bait in this studywas kindly provided byClaus Schwechheimer. Forthe activation-domain vectors, the GAI, RGA, and SPT coding regions wereamplified byPCRwith primers containingSmaI andPstI restriction sites (seeSupplemental Table 1 online). The ampliconswere cloned in frame betweenSmaI and PstI sites of pGAD424 to form the prey vectors. After confirmingthe plasmid quality by sequencing, prey plasmids were transformed intoyeast Y187 (-Leu) and bait plasmids into AH109 (-Trp). Prey transformantswere selected in SD-L medium (SD broth supplemented with 2% Glc[Formedium], complete supplement mixture -Leu [Formedium], pH 5.8, and10% agar) while bait transformants were selected in SD-T medium (SDbroth supplemented with 2% Glc [Formedium], complete supplementmixture -Trp [Formedium], pH 5.8, and 10% agar).

Yeast transformation was performed as described in the ClontechLaboratories Yeast Protocols Handbook, and the desired transformantswere selected on SD-L (prey transformants) or SD-T (bait transformants)media. For each mating combination, a 20-mL aliquot of each of theplasmids was mixed into 160 mL of YPDmedium and incubated overnightat 30°C with shaking at 200 rpm. To confirm mating efficiency andprotein–protein interactions, 10 mL of each mating culture was plated onSD-LT (growth confirms mating) (SD broth supplemented with 2% Glc[Formedium], complete supplement mixture -Leu -Trp [Formedium], pH5.8, and 10% agar) and SD-LTHA plates (growth confirms interaction) (SDbroth supplemented with 2% Glc [Formedium], complete supplementmixture -Ade -His -Leu -Trp [Formedium], pH 5.8, and10% agar) andincubated upside-down at 30°C.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenomeInitiative or GenBank/EMBL databases under the following accessionnumbers:GAI (AT1G14920), RGA (AT2G01570), RGL1 (AT1G66350),RGL2(AT3G03450), RGL3 (AT5G17490), GA1 (AT4G02780), SPT (AT4G36930),GID1A (AT3G05120), GID1B (AT3G63010), GID1C (AT5G27320), SLY1(AT4G24210), TUBULIN8 (AT5G23860), UBIQUITIN-CONJUGATINGENZYME9 (AT4G27960), and UBIQUITIN-CONJUGATING ENZYME10(AT5G5330).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Style Elongation Is Mediated by GA.

Supplemental Figure 2. della Mutants That Do Not Show SignificantParthenocarpy upon Emasculation.

Supplemental Figure 3. Statistical Analysis of the Tissue Sections ofLer, globalMutant, and ga1 global Mutant Pistils as Shown in Figure 3.

Supplemental Figure 4. Function of 26S Proteasome in the DELLA-Independent GA Response in the ga1-3 global-DELLA Mutant Back-ground.

Supplemental Figure 5. SLY1 Is Not Required for the DELLA-Independent GA Response.

Supplemental Figure 6. Characterization of the rgl3-4 Allele.

Supplemental Table 1. Oligonucleotide Sequences.

Gibberellin Signaling in Arabidopsis Fruit Growth 3993

ACKNOWLEDGMENTS

We thank Steve Penfield for giving us access to quad-DELLA spt-2mutant seeds prior to publication of these lines and Claus Schwech-heimer for kindly providing the GID1A-BD vector used as bait in thisstudy. We also thank Andrew Davis from the John Innes Centre forassistance with photography. We thank Nicolas Arnaud, Thomas Girin,and Susana Sauret-Gueto for critically reading the article, Jan Lohmannfor providing useful comments, and Philip Martin for statistical advice.This work was supported by the GRO ISP grant (BB/J004588/1) from theBiotechnology and Biological Sciences Research Council, the JohnInnes Foundation and by a Marie Curie Fellowship on the Early StageTraining Programme MEST-CT-2005-019727 to S.F.

AUTHOR CONTRIBUTIONS

S.F. and L.Ø. designed the research. S.F., K.L., K.S., E.A., and L.Ø.performed the research. S.F., K.L., K.S., E.A., N.P.H., and L.Ø. analyzedthe data. S.F. and L.Ø. wrote the article.

Received July 24, 2012; revised September 17, 2012; acceptedSeptember 24, 2012; published October 12, 2012.

REFERENCES

Achard, P., and Genschik, P. (2009). Releasing the brakes of plant growth:How GAs shutdown DELLA proteins. J. Exp. Bot. 60: 1085–1092.

Achard, P., Gusti, A., Cheminant, S., Alioua, M., Dhondt, S.,Coppens, F., Beemster, G.T., and Genschik, P. (2009). Gibberellinsignaling controls cell proliferation rate in Arabidopsis. Curr. Biol.19: 1188–1193.

Alvarez, J., and Smyth, D.R. (1999). CRABS CLAW and SPATULA,two Arabidopsis genes that control carpel development in parallel

with AGAMOUS. Development 126: 2377–2386.Alvarez, J., and Smyth, D.R. (2002). CRABS CLAW and SPATULA

genes regulate growth and pattern formation during gynoeciumdevelopment in Arabidopsis thaliana. Int. J. Plant Sci. 163: 17–41.

Ariizumi, T., Murase, K., Sun, T.P., and Steber, C.M. (2008). Proteolysis-independent downregulation of DELLA repression in Arabidopsis bythe gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1. Plant

Cell 20: 2447–2459.Arnaud, N., Girin, T., Sorefan, K., Fuentes, S., Wood, T.A.,

Lawrenson, T., Sablowski, R., and Østergaard, L. (2010). Gib-berellins control fruit patterning in Arabidopsis thaliana. Genes Dev.24: 2127–2132.

Bolle, C. (2004). The role of GRAS proteins in plant signal transductionand development. Planta 218: 683–692.

Cao, D., Hussain, A., Cheng, H., and Peng, J. (2005). Loss of function offour DELLA genes leads to light- and gibberellin-independent seedgermination in Arabidopsis. Planta 223: 105–113.

Carbonell-Bejerano, P., Urbez, C., Carbonell, J.J., Granell, A., andPerez-Amador, M.A. (2010). A fertilization-independent developmental

program triggers partial fruit development and senescence processesin pistils of Arabidopsis. Plant Physiol. 154: 163–172.

Chandler, P.M., Marion-Poll, A., Ellis, M., and Gubler, F. (2002).Mutants at the Slender1 locus of barley cv Himalaya. Molecular andphysiological characterization. Plant Physiol. 129: 181–190.

Cheng, H., Qin, L., Lee, S., Fu, X., Richards, D.E., Cao, D., Luo, D.,Harberd, N.P., and Peng, J. (2004). Gibberellin regulates Arabidopsis

floral development via suppression of DELLA protein function. De-velopment 131: 1055–1064.

Chiang, H.H., Hwang, I., and Goodman, H.M. (1995). Isolation of theArabidopsis GA4 locus. Plant Cell 7: 195–201.

Cox, C.M., and Swain, S.M. (2006). Goldacre paper. Localised andnon-localised promotion of fruit development in Arabidopsis. Funct.Plant Biol. 33: 1–8.

Davidonis, G.H. (1990). Gibberellic acid-induced cell elongation incotton suspension-cultures. J. Plant Growth Regul. 9: 243–246.

de Lucas, M., Davière, J.M., Rodríguez-Falcón, M., Pontin, M.,Iglesias-Pedraz, J.M., Lorrain, S., Fankhauser, C., Blázquez,M.A., Titarenko, E., and Prat, S. (2008). A molecular framework for lightand gibberellin control of cell elongation. Nature 451: 480–484.

Del Pozo, J.C., and Estelle, M. (2000). F-box proteins and proteindegradation: An emerging theme in cellular regulation. Plant Mol.Biol. 44: 123–128.

Dill, A., and Sun, T.P. (2001). Synergistic derepression of gibberellinsignaling by removing RGA and GAI function in Arabidopsis thali-ana. Genetics 159: 777–785.

Dill, A., Thomas, S.G., Hu, J., Steber, C.M., and Sun, T.P. (2004).The Arabidopsis F-box protein SLEEPY1 targets gibberellin sig-naling repressors for gibberellin-induced degradation. Plant Cell 16:1392–1405.

Dorcey, E., Urbez, C., Blázquez, M.A., Carbonell, J., and Perez-Amador, M.A. (2009). Fertilization-dependent auxin response inovules triggers fruit development through the modulation of gib-berellin metabolism in Arabidopsis. Plant J. 58: 318–332.

Feng, S., et al. (2008). Coordinated regulation of Arabidopsis thalianadevelopment by light and gibberellins. Nature 451: 475–479.

Ficcadenti, N., Sestili, S., Pandolfini, T., Cirillo, C., Rotino, G.L.,and Spena, A. (1999). Genetic engineering of parthenocarpic fruitdevelopment in tomato. Mol. Breed. 5: 463–470.

Fu, X., Richards, D.E., Ait-Ali, T., Hynes, L.W., Ougham, H., Peng,J., and Harberd, N.P. (2002). Gibberellin-mediated proteasome-dependent degradation of the barley DELLA protein SLN1 re-pressor. Plant Cell 14: 3191–3200.

Fu, X., Richards, D.E., Fleck, B., Xie, D., Burton, N., and Harberd, N.P.(2004). The Arabidopsis mutant sleepy1gar2-1 protein promotes plantgrowth by increasing the affinity of the SCFSLY1 E3 ubiquitin ligase forDELLA protein substrates. Plant Cell 16: 1406–1418.

Gagne, J.M., Downes, B.P., Shiu, S.H., Durski, A.M., and Vierstra,R.D. (2002). The F-box subunit of the SCF E3 complex is encodedby a diverse superfamily of genes in Arabidopsis. Proc. Natl. Acad.Sci. USA 99: 11519–11524.

Gallego-Bartolomé, J., Minguet, E.G., Marín, J.A., Prat, S.,Blázquez, M.A., and Alabadí, D. (2010). Transcriptional di-versification and functional conservation between DELLA proteinsin Arabidopsis. Mol. Biol. Evol. 27: 1247–1256.

Gao, X.H., Xiao, S.L., Yao, Q.F., Wang, Y.J., and Fu, X.D. (2011). Anupdated GA signaling ‘relief of repression’ regulatory model. Mol.Plant 4: 601–606.

Girin, T., Paicu, T., Stephenson, P., Fuentes, S., Körner, E.,O’Brien, M., Sorefan, K., Wood, T.A., Balanzá, V., Ferrándiz, C.,Smyth, D.R., and Østergaard, L. (2011). INDEHISCENT andSPATULA interact to specify carpel and valve margin tissue andthus promote seed dispersal in Arabidopsis. Plant Cell 23: 3641–3653.

Gillaspy, G., Ben-David, H., and Gruissem, W. (1993). Fruits: A de-velopmental perspective. Plant Cell 5: 1439–1451.

Griffiths, J., Murase, K., Rieu, I., Zentella, R., Zhang, Z.L., Powers,S.J., Gong, F., Phillips, A.L., Hedden, P., Sun, T.P., and Thomas,S.G. (2006). Genetic characterization and functional analysis of theGID1 gibberellin receptors in Arabidopsis. Plant Cell 18: 3399–3414.

3994 The Plant Cell

Groszmann, M., Paicu, T., Alvarez, J.P., Swain, S.M., and Smyth, D.R.(2011). SPATULA and ALCATRAZ, are partially redundant, functionallydiverging bHLH genes required for Arabidopsis gynoecium and fruitdevelopment. Plant J. 68: 816–829.

Gustafson, F.G. (1936). Inducement of fruit development by growth-promoting chemicals. Proc. Natl. Acad. Sci. USA 22: 628–636.

Harberd, N.P. (2003). Botany. Relieving DELLA restraint. Science 299:1853–1854.

Hedden, P., and Phillips, A.L. (2000). Gibberellin metabolism: Newinsights revealed by the genes. Trends Plant Sci. 5: 523–530.

Heisler, M.G., Atkinson, A., Bylstra, Y.H., Walsh, R., and Smyth, D.R.(2001). SPATULA, a gene that controls development of carpel margintissues in Arabidopsis, encodes a bHLH protein. Development 128:1089–1098.

Ichihashi, Y., Horiguchi, G., Gleissberg, S., and Tsukaya, H. (2010).The bHLH transcription factor SPATULA controls final leaf size inArabidopsis thaliana. Plant Cell Physiol. 51: 252–261.

Inada, S., and Shimmen, T. (2000). Regulation of elongation growthby gibberellin in root segments of Lemna minor. Plant Cell Physiol.41: 932–939.

Josse, E.M., Gan, Y., Bou-Torrent, J., Stewart, K.L., Gilday, A.D.,Jeffree, C.E., Vaistij, F.E., Martínez-García, J.F., Nagy, F.,Graham, I.A., and Halliday, K.J. (2011). A DELLA in disguise:SPATULA restrains the growth of the developing Arabidopsisseedling. Plant Cell 23: 1337–1351.

King, G.N. (1947). Artificial parthenocarpy in Lycopersicum escu-lentum; Tissue development. Plant Physiol. 22: 572–581.

King, K.E., Moritz, T., and Harberd, N.P. (2001). Gibberellins are notrequired for normal stem growth in Arabidopsis thaliana in the ab-sence of GAI and RGA. Genetics 159: 767–776.

Koini, M.A., Alvey, L., Allen, T., Tilley, C.A., Harberd, N.P.,Whitelam, G.C., and Franklin, K.A. (2009). High temperature-mediated adaptations in plant architecture require the bHLH tran-scription factor PIF4. Curr. Biol. 19: 408–413.

Lee, S., Cheng, H., King, K.E., Wang, W., He, Y., Hussain, A., Lo, J.,Harberd, N.P., and Peng, J. (2002). Gibberellin regulates Arabidopsisseed germination via RGL2, a GAI/RGA-like gene whose expression isup-regulated following imbibition. Genes Dev. 16: 646–658.

Magnus, V., Ozga, J.A., Reinecke, D.M., Pierson, G.L., Larue, T.A.,Cohen, J.D., and Brenner, M.L. (1997). 4-Chloroindole-3-aceticacid and indole-3-acetic acid in Pisum sativum. Phytochemistry 46:675–681.

Martí, C., Orzáez, D., Ellul, P., Moreno, V., Carbonell, J., andGranell, A. (2007). Silencing of DELLA induces facultative parthe-nocarpy in tomato fruits. Plant J. 52: 865–876.

McGinnis, K.M., Thomas, S.G., Soule, J.D., Strader, L.C., Zale, J.M., Sun, T.P., and Steber, C.M. (2003). The Arabidopsis SLEEPY1gene encodes a putative F-box subunit of an SCF E3 ubiquitin li-gase. Plant Cell 15: 1120–1130.

Mezzetti, B., Landi, L., Pandolfini, T., and Spena, A. (2004). ThedefH9-iaaM auxin-synthesizing gene increases plant fecundity andfruit production in strawberry and raspberry. BMC Biotechnol. 4: 4.

Murase, K., Hirano, Y., Sun, T.P., and Hakoshima, T. (2008). Gibberellin-induced DELLA recognition by the gibberellin receptor GID1. Nature 456:459–463.

Nakajima, M., et al. (2006). Identification and characterization ofArabidopsis gibberellin receptors. Plant J. 46: 880–889.

Ngo, P., Ozga, J.A., and Reinecke, D.M. (2002). Specificity of auxinregulation of gibberellin 20-oxidase gene expression in pea peri-carp. Plant Mol. Biol. 49: 439–448.

Oh, E., Yamaguchi, S., Hu, J., Yusuke, J., Jung, B., Paik, I., Lee, H.S.,Sun, T.P., Kamiya, Y., and Choi, G. (2007). PIL5, a phytochrome-interacting bHLH protein, regulates gibberellin responsiveness by

binding directly to the GAI and RGA promoters in Arabidopsis seeds.Plant Cell 19: 1192–1208.

Olszewski, N., Sun, T.P., and Gubler, F. (2002). Gibberellin signaling:Biosynthesis, catabolism, and response pathways. Plant Cell 14(Suppl): S61–S80.

Ozga, J.A., and Reinecke, D.M. (2003). Hormonal interactions in fruitdevelopment. J. Plant Growth Regul. 22: 73–81.

Ozga, J.A., Reinecke, D.M., Ayele, B.T., Ngo, P., Nadeau, C., andWickramarathna, A.D. (2009). Developmental and hormonal reg-ulation of gibberellin biosynthesis and catabolism in pea fruit. PlantPhysiol. 150: 448–462.

Ozga, J.A., van Huizen, R., and Reinecke, D.M. (2002). Hormoneand seed-specific regulation of pea fruit growth. Plant Physiol. 128:1379–1389.

Penfield, S., Josse, E.M., Kannangara, R., Gilday, A.D., Halliday, K.J., and Graham, I.A. (2005). Cold and light control seed germina-tion through the bHLH transcription factor SPATULA. Curr. Biol. 15:1998–2006.

Peng, J., Carol, P., Richards, D.E., King, K.E., Cowling, R.J.,Murphy, G.P., and Harberd, N.P. (1997). The Arabidopsis GAI genedefines a signaling pathway that negatively regulates gibberellinresponses. Genes Dev. 11: 3194–3205.

Peng, J., Richards, D.E., Moritz, T., Ezura, H., Carol, P., andHarberd, N.P. (2002). Molecular and physiological characterizationof Arabidopsis GAI alleles obtained in targeted Ds-tagging experi-ments. Planta 214: 591–596.

Phillips, A.L., Ward, D.A., Uknes, S., Appleford, N.E., Lange, T.,Huttly, A.K., Gaskin, P., Graebe, J.E., and Hedden, P. (1995).Isolation and expression of three gibberellin 20-oxidase cDNAclones from Arabidopsis. Plant Physiol. 108: 1049–1057.

Rieu, I., Eriksson, S., Powers, S.J., Gong, F., Griffiths, J., Woolley,L., Benlloch, R., Nilsson, O., Thomas, S.G., Hedden, P., andPhillips, A.L. (2008). Genetic analysis reveals that C19-GA 2-oxi-dation is a major gibberellin inactivation pathway in Arabidopsis.Plant Cell 20: 2420–2436.

Roeder, A.H.K., and Yanofsky, M.F. (2006). Fruit development inArabidopsis. The Arabidopsis Book 4: e0075, doi/.10.1199/tab.0075

Ross, J.J., Reid, J.B., Murfet, I.C., and Weston, D.E. (2008). Theslender phenotype of pea is deficient in DELLA proteins. PlantSignal. Behav. 3: 590–592.

Rotino, G.L., Perri, E., Zottini, M., Sommer, H., and Spena, A.(1997). Genetic engineering of parthenocarpic plants. Nat. Bio-technol. 15: 1398–1401.

Sanchez-Fernandez, R., Ardiles-Diaz, W., Van Montagu, M., Inze,D., and May, M.J. (1998). Cloning of a novel Arabidopsis thalianaRGA-like gene, a putative member of the VHIID-domain transcrip-tion factor family. J. Exp. Bot. 49: 1609–1610.

Santner, A., and Estelle, M. (2009). Recent advances and emergingtrends in plant hormone signalling. Nature 459: 1071–1078.

Sasaki, A., Itoh, H., Gomi, K., Ueguchi-Tanaka, M., Ishiyama, K.,Kobayashi, M., Jeong, D.H., An, G., Kitano, H., Ashikari, M., andMatsuoka, M. (2003). Accumulation of phosphorylated repressorfor gibberellin signaling in an F-box mutant. Science 299: 1896–1898.

Serrani, J.C., Ruiz-Rivero, O., Fos, M., and García-Martínez, J.L.(2008). Auxin-induced fruit-set in tomato is mediated in part bygibberellins. Plant J. 56: 922–934.

Sidaway-Lee, K., Josse, E.M., Brown, A., Gan, Y., Halliday, K.J.,Graham, I.A., and Penfield, S. (2010). SPATULA links daytimetemperature and plant growth rate. Curr. Biol. 20: 1493–1497.

Silverstone, A.L., Ciampaglio, C.N., and Sun, T. (1998). The Arabi-dopsis RGA gene encodes a transcriptional regulator repressing thegibberellin signal transduction pathway. Plant Cell 10: 155–169.

Gibberellin Signaling in Arabidopsis Fruit Growth 3995

Silverstone, A.L., Jung, H.S., Dill, A., Kawaide, H., Kamiya, Y., and Sun,T.P. (2001). Repressing a repressor: Gibberellin-induced rapid reductionof the RGA protein in Arabidopsis. Plant Cell 13: 1555–1566.

Smyth, D.R., Bowman, J.L., and Meyerowitz, E.M. (1990). Earlyflower development in Arabidopsis. Plant Cell 2: 755–767.

Sorefan, K., Girin, T., Liljegren, S.J., Ljung, K., Robles, P., Galván-Ampudia, C.S., Offringa, R., Friml, J., Yanofsky, M.F., andØstergaard, L. (2009). A regulated auxin minimum is required forseed dispersal in Arabidopsis. Nature 459: 583–586.

Srinivasan, A., and Morgan, D.G. (1996). Growth and development ofthe pod wall in spring rape (Brassica napus) as related to thepresence of seeds and exogenous phytohormones. J. Agric. Sci.127: 487–500.

Srivastava, L.M., Sawhney, V.K., and Taylor, I.E. (1975). Gibberellic-acid-induced cell elongation in lettuce hypocotyls. Proc. Natl. Acad.Sci. USA 72: 1107–1111.

Sun, T., Goodman, H.M., and Ausubel, F.M. (1992). Cloning theArabidopsis GA1 locus by genomic subtraction. Plant Cell 4:119–128.

Sun, T.P. (2011). The molecular mechanism and evolution of the GA-GID1-DELLA signaling module in plants. Curr. Biol. 21: R338–R345.

Talon, M., Zacarias, L., and Primo-Millo, E. (1990). Hormonalchanges associated with fruit set and development in mandarinsdiffering in their parthenocarpic ability. Plant Physiol. 79: 400–406.

Thomas, S.G., Phillips, A.L., and Hedden, P. (1999). Molecularcloning and functional expression of gibberellin 2- oxidases, mul-tifunctional enzymes involved in gibberellin deactivation. Proc. Natl.Acad. Sci. USA 96: 4698–4703.

Tyler, L., Thomas, S.G., Hu, J., Dill, A., Alonso, J.M., Ecker, J.R.,and Sun, T.P. (2004). Della proteins and gibberellin-regulated seedgermination and floral development in Arabidopsis. Plant Physiol.135: 1008–1019.

Ubeda-Tomás, S., Federici, F., Casimiro, I., Beemster, G.T.,Bhalerao, R., Swarup, R., Doerner, P., Haseloff, J., and Bennett,M.J. (2009). Gibberellin signaling in the endodermis controls Arab-idopsis root meristem size. Curr. Biol. 19: 1194–1199.

Ubeda-Tomás, S., Swarup, R., Coates, J., Swarup, K., Laplaze, L.,Beemster, G.T., Hedden, P., Bhalerao, R., and Bennett, M.J.

(2008). Root growth in Arabidopsis requires gibberellin/DELLA sig-nalling in the endodermis. Nat. Cell Biol. 10: 625–628.

Ueguchi-Tanaka, M., Ashikari, M., Nakajima, M., Itoh, H., Katoh, E.,Kobayashi, M., Chow, T.Y., Hsing, Y.I., Kitano, H., Yamaguchi, I.,and Matsuoka, M. (2005). GIBBERELLIN INSENSITIVE DWARF1encodes a soluble receptor for gibberellin. Nature 437: 693–698.

Ueguchi-Tanaka, M., Nakajima, M., Katoh, E., Ohmiya, H., Asano,K., Saji, S., Hongyu, X., Ashikari, M., Kitano, H., Yamaguchi, I.,and Matsuoka, M. (2007). Molecular interactions of a soluble gib-berellin receptor, GID1, with a rice DELLA protein, SLR1, and gib-berellin. Plant Cell 19: 2140–2155.

van Huizen, R., Ozga, J.A., Reinecke, D.M., Twitchin, B., andMander, L.N. (1995). Seed and 4-chloroindole-3-acetic acid regu-lation of gibberellin metabolism in pea pericarp. Plant Physiol. 109:1213–1217.

Vivian-Smith, A., and Koltunow, A.M. (1999). Genetic analysis ofgrowth-regulator-induced parthenocarpy in Arabidopsis. PlantPhysiol. 121: 437–451.

Vivian-Smith, A., Luo, M., Chaudhury, A., and Koltunow, A.M.(2001). Fruit development is actively restricted in the absence offertilization in Arabidopsis. Development 128: 2321–2331.

Wen, C.K., and Chang, C. (2002). Arabidopsis RGL1 encodes a neg-ative regulator of gibberellin responses. Plant Cell 14: 87–100.

Willige, B.C., Ghosh, S., Nill, C., Zourelidou, M., Dohmann, E.M., Maier,A., and Schwechheimer, C. (2007). The DELLA domain of GAINSENSITIVE mediates the interaction with the GA INSENSITIVEDWARF1A gibberellin receptor of Arabidopsis. Plant Cell 19: 1209–1220.

Wittwer, S.H., Bukovac, M.J., Sell, H.M., and Weller, L.E. (1957).Some effects of gibberellin on flowering and fruit setting. PlantPhysiol. 32: 39–41.

Xu, Y.L., Li, L., Wu, K., Peeters, A.J., Gage, D.A., and Zeevaart, J.A.(1995). The GA5 locus of Arabidopsis thaliana encodes a multi-functional gibberellin 20-oxidase: Molecular cloning and functionalexpression. Proc. Natl. Acad. Sci. USA 92: 6640–6644.

Zentella, R., Zhang, Z.L., Park, M., Thomas, S.G., Endo, A.,Murase, K., Fleet, C.M., Jikumaru, Y., Nambara, E., Kamiya, Y.,and Sun, T.P. (2007). Global analysis of della direct targets in earlygibberellin signaling in Arabidopsis. Plant Cell 19: 3037–3057.

3996 The Plant Cell

DOI 10.1105/tpc.112.103192; originally published online October 12, 2012; 2012;24;3982-3996Plant Cell

Sara Fuentes, Karin Ljung, Karim Sorefan, Elizabeth Alvey, Nicholas P. Harberd and Lars ØstergaardResponses

Occurs via DELLA-Dependent and DELLA-Independent GibberellinArabidopsisFruit Growth in

 This information is current as of May 4, 2018

 

Supplemental Data /content/suppl/2012/10/09/tpc.112.103192.DC1.html

References /content/24/10/3982.full.html#ref-list-1

This article cites 89 articles, 45 of which can be accessed free at:

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists