ORIGINAL PAPER

Characterisation of the DELLA subfamily in apple(Malus x domestica Borkh.)

Toshi Foster & Chris Kirk & William T. Jones &

Andrew C. Allan & Richard Espley &

Sakuntala Karunairetnam & Jasna Rakonjac

Received: 19 January 2006 /Revised: 12 May 2006 /Accepted: 24 May 2006 /Published online: 17 November 2006# Springer-Verlag 2006

Abstract The hormone gibberellic acid (GA) regulatesgrowth and development throughout the plant life cycle.DELLA proteins are key components of the GA signallingpathway and act to repress GA responses. The “DELLA”amino acid motif is highly conserved among diversespecies and is essential for GA-induced destruction ofDELLA proteins, which relieves repression. Six genesencoding the DELLA motif were identified within an appleexpressed sequence tag (EST) database. Full-length cDNAclones were obtained by RACE and these were designatedMdRGL1a/b, MdRGL2a/b, and MdRGL3a/b. Sequencealignment of the predicted proteins indicates that theMdDELLAs are 37–93% homologous to one another and44–65% to the Arabidopsis DELLAs. The MdDELLAscluster into three pairs, which reflect the presumedallopolyploid origins of the Maloideae. Expression analysisusing quantitative real-time PCR indicates that all threepairs of MdDELLA mRNAs are expressed at the highestlevels in summer arrested shoot tips and in autumnvegetative buds. Transgenic Arabidopsis expressingMdRGL2a have smaller leaves and shorter stems, take

longer to flower in short days, and exhibit a reducedresponse to exogenous GA3, indicating significant conser-vation of gene function between DELLA proteins fromapple and Arabidopsis.

Keywords Apple . DELLA proteins . Flowering .

Growth termination . Gibberellin (GA) . GAI/RGA

Introduction

Gibberellins (GAs) are phytohormones that promote im-portant aspects of growth such as seed germination, leafexpansion, stem elongation, flowering, and fruit develop-ment (Davies 1985). While the GA biosynthetic pathway iswell characterised, the GA signal transduction pathway isstill being elucidated (Alvey and Harberd 2005; Fleet andSun 2005; Hedden and Phillips 2000). Mutants that makebioactive GAs but show an altered GA response have beeninvaluable in identifying genes involved in GA signalling(Hooley 1994; Swain and Olszewski 1996; Ueguchi-Tanaka et al. 2005).

Semi-dominant, GA-insensitive dwarf mutants havebeen identified in a number of species (Chandler et al.2002; Gale et al. 1975; Harberd and Freeling 1989; Ikeda etal. 2001; Koorneef et al. 1985; Skene and Barlass 1983).The gene conferring the Arabidopsis GA-Insensitive (gai)dwarf mutant phenotype was the first to be cloned (Peng etal. 1997). The GAI protein has a high degree of C-terminalhomology with a group of plant-specific, putative tran-scription regulators now known as the GRAS family (Bolle2004; Pysh et al. 1999; Tian et al. 2004). GRAS proteins,named after the first characterised members, GAI, RGA(Repressor of ga1-3), and SCR, are involved with multipleaspects of plant growth and development. The mutant gai

Tree Genet Genomes (2007) 3:187–197DOI 10.1007/s11295-006-0047-z

Electronic supplementary material Supplementary material isavailable for this article at http://dx.doi.org/10.1007/s11295-006-0047-zand is accessible for authorized users.

T. Foster (*) : C. Kirk :W. T. JonesThe Horticulture and Food Research Institute of New Zealand Ltd,PB 11030, Palmerston North, New Zealande-mail: tfoster@hortresearch.co.nz

C. Kirk :A. C. Allan : R. Espley : S. KarunairetnamThe Horticulture and Food Research Institute of New Zealand Ltd,PB 92169, Auckland, New Zealand

J. RakonjacInstitute of Molecular Biosciences, Massey University,PB 11222, Palmerston North, New Zealand

gene carries an N-terminal deletion of 17 amino acids thatalters the activity of the protein (Peng et al. 1997). Thedeleted region begins with the amino acids DELLA,the motif which distinguishes the DELLA subfamily. TheArabidopsis genome encodes four other DELLA proteins:RGA, RGL1, RGL2 and RGL3 (RGA-like1/2/3) (Dill et al.2001; Lee et al. 2002; Silverstone et al. 1998). The DELLAproteins have unique and overlapping functions as negativeregulators of GA response (Lee et al. 2002; Tyler et al.2004; Wen and Chang 2002).

DELLA proteins function analogously in rice (SLR1),wheat (Rht-B1b, Rht-D1b), maize (D8), barley (SLN1) andgrape (VvGAI) (Boss and Thomas 2002; Chandler et al.2002; Ogawa et al. 2000; Peng et al. 1999). For example,the semi-dominant dwarf wheat mutants that were the basisof the ‘Green Revolution’ cultivars are caused by mutationto the DELLA region of the Rht-B1/Rht-D1 genes (Peng etal. 1999). The highly productive, semi-dwarfed grapecultivar Pinot Meunoir contains a single amino acid changeto the DELLA domain of VvGAI (Boss and Thomas 2002).In diverse species, mutation to the DELLA region results ina GA-insensitive dominant dwarf phenotype, indicating thatthe function of the DELLA motif in the response to GA ishighly conserved (Chandler et al. 2002; Dill et al. 2001;Hynes et al. 2003; Itoh et al. 2002; Peng et al. 1999).

DELLA proteins are localised to the nucleus, consistentwith their presumed function as transcriptional regulators(Fleck and Harberd 2002; Gubler et al. 2002; Ogawa et al.2000; Silverstone et al. 1998). Experiments have demon-strated that GA induces the rapid disappearance of RGA,SLR1 and SLN1 from the nucleus. Deletion of the DELLAmotif renders each of these proteins resistant to GA-induceddegradation and causes a GA-insensitive phenotype whenexpressed in plants (Chandler et al. 2002; Dill et al. 2001;Gubler et al. 2002; Itoh et al. 2002; Silverstone et al. 2001).These findings suggest that GA-induced DELLA degrada-tion is the biochemical basis of DELLA function in GAsignalling, corroborating the genetic data. Recently, it hasbeen demonstrated that DELLA proteins are destroyed via aSCF E3 ubiquitin ligase pathway (Dill et al. 2004; Fu et al.2002, 2004; Sasaki et al. 2003). Together, the data support amodel in which the DELLA proteins act as negativeregulators and GA overcomes their action by targetingthem for destruction.

Because many GA-mediated processes are agriculturallyimportant, GA levels are routinely manipulated by chemicalmeans to attain desirable effects in a number of species(Looney 1997; Rademacher 2000). For example, exoge-nous GA is used to control flowering time and increase fruitsize, and inhibitors of GA biosynthesis are applied torestrict vegetative growth (Black 2004; Greene 1989,1999). Apple (Malus x domestica Borkh.) is one of themost economically important tree crops grown worldwide.

Commercial apple growers use a combination of chemicalapplication, manual fruit thinning and tree pruning tocontrol flowering, fruit growth and canopy architecture(Ferree and Warrington 2003). To investigate the role ofDELLA proteins in these processes, we have identifiedDELLA-encoding genes from apple. There is potential toreduce chemical use and labour-intensive practices incommercial apple production by introducing genetic var-iants of MdDELLAs into new cultivars.

In this paper, we describe the isolation of six MdDELLAsand characterise gene expression patterns by quantitativereal-time PCR. We show that the overexpression ofMdRGL2a in Arabidopsis is able to profoundly altervegetative development, flowering time in short days, andresponsiveness to exogenous GA, indicating functionalactivity of orthologous genes.

Materials and methods

Gene cloning and sequence analysis

Two highly conserved N-terminal motifs (DELLA,TVHYNP) and the conserved C-terminal portion ofArabidopsis DELLA proteins were used to mine an appleEST database containing more than 164,000 ESTs forMalusdomestica orthologues of GAI/RGA. Six different DELLA-encoding genes were identified, and three of these weretruncated at either the 5′ or 3′ end. Missing ends of cDNAswere obtained using rapid amplified cDNA ends (RACE)according to manufacturer’s instructions (Invitrogen).mRNA for RACE was extracted from Royal Gala shoottips using poly dT beads (Dynal). The gene-specific RACEprimers are listed in Supplementary Table 1. The differ-ences between the existing sequences of the six genes werehigh enough to design gene-specific primers. In the caseswhere the primer was conserved between a/b gene pairs,there was enough overlap with the pre-existing sequence todistinguish between the ‘a’ and ‘b’ RACE products.

Total RNA was extracted from Royal Gala shoot tipsusing an RNeasy Plant Mini kit (Qaigen, Valencia, CA,USA). First-strand cDNA was synthesised from 1 μg ofRNA, using Moloney Murine Leukaemia Virus (M-MuLV)reverse transcriptase and a poly dT primer for one h at 37°C(Amersham-GE Healthcare). Full-length cDNAs were thenamplified from first-strand cDNA using HiFi Taq polymer-ase (Invitrogen) and gene-specific 5′/3′ primers based on the5′/3′ RACE products. Products were cloned using a TAcloning kit (Invitrogen).

Sequence data was edited using Vector NTI and multipleamino acid alignments of the predicted proteins wereconstructed using CLUSTAL X. The alignment parametersused were: gap open penalty 10; gap extension penalty 0.1;

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40% identity for alignment delay; BLOSUM series matrix;residue-specific gaps on and hydrophylic residue gap on.Phylogenetic analysis was performed using PHYLYP 3.63.Neighbour-joining distance criteria were used for phyloge-netic estimation. The statistical reliability of the phylogenetictree was tested by bootstrap analysis with 500 replicates. TheGenBank accession numbers are: MdRGL2a, DQ007883;MdRGL2b, DQ007884; MdRGL1a, DQ007885; MdRGL1b,DQ007886; MdRGL3a, DQ007887; and MdRGL3b,DQ007888.

Plant material and expression analysis using quantitativereal-time PCR

Shoot apex and leaf samples were collected from matureRoyal Gala and Pacific Rose trees growing at theHortResearch field station near Havelock North, NewZealand. All samples were collected at midday from well-exposed positions. Leaves larger than 5 mm and/or anybudscales were removed from shoot tip and bud samples.Total RNA was extracted from 100 mg of the followingRoyal Gala tissues: 1) apices of spur-type shoots that hadinitiated budscales and arrested vegetative growth, 2) apicesof extension shoots still initiating vegetative leaves, 3)leaves 5–10 mm in length, 4) seeds vernalised for 8 weeksat 4°, then imbibed on moist filter paper for 3 days, or 5)eight days. Under New Zealand growing conditions, mostRoyal Gala terminal buds are floral. To obtain terminalvegetative buds, RNAwas also extracted from 6) vegetativeand 7) floral buds from Pacific Rose, a cultivar withpronounced biennial bearing (alternate floral and vegetativeyears). Samples 1–3 were collected in early summer(9.12.03), 53 days after full bloom (DAFB), and samples6 and 7 were collected in the autumn after fruit harvest(29.04.04), 193 DAFB.

Total RNA extraction and first-strand cDNA synthesiswere performed as described above. One twentieth (1/20) ofthe cDNA sample was used as a template for the real-timePCR, using the LightCycler fast start DNA master SYBR Igreen kit in the LightCycler real-time thermocycler (Roche).Gene-specific primers for detecting transcripts ofMdRGL1a,MdRGL1b, MdRGL2a, MdRGL2b, MdRGL3a, MdRGL3band MdGAPDH are listed in Supplemental Table 1. Primerswere used at 1.0 μM each in a 10 μl reaction volume. PCRparameters were: initial denaturation at 95°C for 10 minthen 40 cycles of 95°C 10 s, 68°C 5 s, 72°C 10 s (annealingtemperature was 60°C for MdGAPDH primers). Each PCRproduct was analysed by agarose gel electrophoresis andthe melting curve was determined to verify the presence ofa single product of the correct size.

As a standard for calibration, a series of five tenfolddilutions of chromosomal DNAwas used as a template. Thecopy number of the gene of interest in the standard was

estimated to be 1.3×103/ng based on the estimation that thegenome size of M. domestica is 770 Mbp, approximatelysix times that of A. thaliana (Patocchi et al. 1999). EverymRNA of interest was quantified by a separate experiment.Each quantification experiment consisted of 13 reactions(seven cDNA samples, five standards and a negativecontrol without template). To eliminate variation due tothe differences in total mRNA amount in the samples, theMdDELLA mRNA copy number was normalised to that ofMdGAPDH, which is relatively constant in all tissue typesand developmental stages (Iskandar et al. 2004). Prelimi-nary analysis indicated that the expression level of the a/bgene pairs was very similar. Due to the limited amount ofRNA samples, quantification of the ‘b’ gene was carriedout in triplicate and presented in Fig. 3.

Plant transformation and analysis

The full-length MdRGL2a cDNA was cloned into thepART27 plant transformation vector, containing the CaMV35S promoter, octopine synthase terminator, and kanamycinresistance as selection marker (Gleave 1992). Agrobacteriumtumifaciens strain GV3101 was used for floral dip transfor-mation of Arabidopsis (Columbia ecotype) (Clough andBent 1998). Resultant seeds were plated on 0.5X MS culturecontaining 50 μg/ml kanamycin to select for T1 trans-formants. Six independent kanamycin resistant transgenicplants were recovered and five produced viable progeny.Total RNA was extracted from leaves of mature plants andfirst-strand cDNA synthesis was performed as describedabove. Transgene expression levels were analysed byquantitative real-time PCR using MdRGL2a-specific primersas described above. In this experiment, MdRGL2a cDNAlevels were normalised relative to Atactin cDNA (Supple-mentary Table 1). Phenotypic analysis was performed on T3

and subsequent generations. For analysis of leaf size andplant height, plants were grown for 25 days in long-dayconditions (16 h light/8 h dark), one plant per pot. Plantheight was measured along the primary inflorescence axisand the maximum rosette diameter was measured for 10–20plants per line. Plants were grown in 8 h light/16 h dark todetermine time to flowering in short-day (SD) conditions.Flowering was scored when petals were first visible. Most ofthe plants expressing MdRGL2a had not flowered by140 days in SD, so the data are presented as percentflowering by 94 days in SD.

Seeds from transgenic lines 1, 2 and 5, and the emptyvector control were vernalised at 4°C for 4 days, sterilised,rinsed, then plated onto 0.5X MS media (controls) or mediacontaining 10−4 M GA3 (Valent Biosciences, USA). Seed-lings were grown in a growth room maintained at 20°C witha 16 h light/8 h dark cycle of fluorescent light for 7 days.Hypocotyls growing upright and not in contact with

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anything else were selected for measurement. Seedlings wereplaced in a drop of water on a glass slide and hypocotyllength (from the cotyledon shoulders to the collet of roothairs) was measured from calibrated digital images (LeicaMZFLIII stereomicroscope equipped with a DC200 digitalcamera; Wetzlar, Germany). This experiment was replicatedthree times with similar results. Figure 5 shows data fromone experiment.

Results

Cloning and sequence analysis of the MdDELLAs

Six DELLA-encoding genes were identified from an appleEST database containing more than 164,000 sequences(Newcomb et al. 2006). Three were full-length cDNAs andthe remaining three were truncated at either the 5′ or 3′ end.The missing ends were obtained by RACE, and full-lengthcDNAs were amplified from Royal Gala cDNA usingprimers based on 5′ or 3′ RACE products. The six genescluster into three pairs and were designated MdRGL1a,MdRGL1b, MdRGL2a, MdRGL2b, MdRGL3a andMdRGL3b, for Malus domestica RGA-like (GenBankaccession numbers DQ007883–DQ007888). Each a/b pairshares 91–93% homology at the amino acid level, reflectingthe presumed allopolyploid origins of the Maloideae(Evans and Campbell 2002). The homologous pairs aremore divergent from one another than they are to the sameallele from different cultivars, which tend to be 98–99%homologous to one another (data not shown). TheMdRGL1a/b, MdRGL2a/b and MdRGL3a/b ORFs are 1.9,1.7 and 1.6 kb, respectively. Based on PCR analysis ofgenomic DNA, none of these genes contain introns, whichis consistent with DELLA-encoding genes from other plantspecies (data not shown). We refer to the group of six genesand proteins as MdDELLAs and MdDELLAs, respectively.

The predicted molecular mass of MdRGL1a/b,MdRGL2a/b and MdRGL3a/b proteins are 70, 64 and60 kD, respectively. A multiple alignment indicates that theN-termini of these proteins have the two signature motifs,DELLA and TVHYNP, which define the DELLA subfam-ily and are necessary for GA-induced degradation ofDELLA proteins (Fig. 1). MdRGL3a/b diverge from theconsensus sequence within the DELLA domain withsubstitutions to 7/27 amino acids. The C-termini of theMdDELLAs have five highly conserved motifs (LHRI,VHIID, LHRII, PFYRE and SAW) that are shared by thelarger family of GRAS proteins.

Overall, these proteins share 37–38% (MdRGL1a/b vsMdRGL3a/b), 44–46% (MdRGL2a/b vs MdRGL3a/b), and61–64% (MdRGL2a/b vs MdRGL1a/b) homology to oneanother. The MdDELLAs are most divergent over their

N-termini, and are highly homologous over their C-termini.Alignment with Arabidopsis DELLA proteins showed thatMdRGL1a/b and MdRGL2a/b are 62–63% homologous toGAI and RGA. MdRGL3a/b are the most divergent,sharing 45–51% homology with all of the ArabidopsisDELLA proteins. Phylogenetic analysis indicates thatMdRGL3a/b are more closely related to DELLA proteinsfrom monocots (Fig. 2). Sequence homology to otherDELLA proteins is slightly higher for the proteins encodedby the ‘b’ gene of all three pairs of MdDELLAs.

Expression analysis of MdDELLAs

Real-time PCR with gene-specific primers was used toquantify transcript levels of individual MdDELLAs indifferent apple tissues of various developmental stages.First-strand cDNA was reverse-transcribed from total RNAand used as a template in PCR reactions. The cDNA copynumber of each gene is represented relative to that ofMdGAPDH, a constitutively expressed housekeeping gene(Iskandar et al. 2004). All six MdDELLAs are transcription-ally active, although some are expressed at very low levels(cDNA copies ≤0.01 that of MdGAPDH). There was verylittle variation in expression between homologous pairs ofMdDELLA genes (data not shown), therefore, expressionanalysis of only the ‘b’ gene is represented in Fig. 3.

In early summer, spur-type shoots cease leaf initiation andenter a period of developmental arrest, whereas extensionshoots continue to initiate leaves and undergo internodeextension for another 4–8 weeks (Fulford 1965, 1966). Inspur-type shoot apices that have recently arrested growth,MdRGL3 is expressed at a much higher level (0.193 that ofMdGAPDH cDNA) than are MdRGL1 and MdRGL2 (0.082and 0.048, respectively). At this same time-point, all sixMdDELLAs are expressed at very low levels in the apices ofactively growing shoots and in young expanding leaves(≤0.01). This data is consistent with the current understand-ing of DELLAs as repressors of growth and would suggest adominant role for MdRGL3 in initiating and/or maintainingdevelopmental arrest of the meristem.

By late summer, all terminal buds have either arrested asvegetative meristems, or have undergone floral develop-ment (Foster et al. 2003). In autumn, all six MdDELLAs areexpressed at high levels in vegetative buds (0.165–0.213)and moderately low levels in floral buds (0.018–0.032).MdDELLA transcripts, MdRGL2 in particular, are muchmore abundant in floral buds than in expanding leaves andgrowing apices. Based on these expression profiles, wesuggest that all six MdDELLAs are involved with restrictinggrowth in autumn buds, but may play a greater role invegetative buds than in floral buds.

After 8 weeks vernalisation at 4°C, Royal Gala seedswere germinated on moist filter paper. There is a slight

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increase in both MdRGL1 and MdRGL2 expression be-tween 3 and 8 days of germination. However, theMdDELLAs are all expressed at very low levels ingerminating seeds (0.003–0.012), unlike ArabidopsisRGL1/2/3, which are expressed at very high levels duringgermination (Lee et al. 2002; Tyler et al. 2004). Thisdiscrepancy could be due to transient expression fromunstable MdDELLA mRNAs, or the six MdDELLAscharacterised in this paper may not be involved inregulating seed germination.

Expression of MdRGL2a in Arabidopsis

To determine if MdDELLAs function analogously to DELLAproteins in Arabidopsis, a binary vector containing the 35S

CaMV promoter driving the full-length MdRGL2a cDNAwastransformed into Arabidopsis using the floral dip method(Clough and Bent 1998). MdRGL2a was selected for over-expression analysis because it is intermediate betweenMdRGL1a/b and MdRGL3a/b in terms of deviation from theconsensus sequence within the DELLA motif and the down-regulation of MdRGL2 expression in growing shoots andexpanding leaves is the most pronounced among theMdDELLAs (Fig. 3) Six independent kanamycin-resistanttransgenic plants were generated, but only five of the primarytransformants produced progeny. Transgene expression levelswere determined by quantitative real-time PCR and werenormalised against Atactin cDNA (Fig. 4a). Compared toempty vector controls, plants expressing MdRGL2a haveshortened inflorescence lengths and smaller leaves (Fig. 4b,c).

Fig. 1 Amino acid sequence alignment of MdRGL1a/b, MdRGL2a/band MdRGL3a/b. Gaps introduced to maximise alignment are indicatedby dashes. Conserved motifs are indicated by overhead lines. A nuclear

localisation signal (NLS), indicated by a dashed line, lies within the firstleucine heptad repeat (LHRI). Conserved sequences in PFYRE andRVER/SAW motifs are indicated by solid lines

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p35S:MdRGL2a plants grown in short days were significantlydelayed in flowering relative to empty vector controls, withmost plants taking longer than 140 days to flower (Fig. 4d).These phenotypes vary from mild to severe and are correlatedwith the level of MdRGL2a expression in each line.

Transgenic overexpression of DELLA-encoding genes hasbeen shown to reduce the response to exogenous GA (Ait-ali etal. 2003; Fleck and Harberd 2002; Fu et al. 2001; Hynes et al.2003; Itoh et al. 2002). To determine if the overexpression ofMdRGL2a confers a similar response, we measured the effectexogenous GA3 on seedling hypocotyl elongation in the threelines showing the most severe phenotypes (1, 2 and 5) and theempty vector control (C). After 7 days of growth, controlseedlings grown on 10−4 M GA3 showed a 1.6-fold increasein hypocotyl length over untreated seedlings (Fig. 5). In the

absence of exogenous GA3, the hypocotyls of p35S:MdRGL2a seedlings (1, 2, and 5) were significantly shorterthan those of the controls (Fig. 5). Lines 1, 2 and 5 did notrespond to GA treatment to the same extent as controlseedlings, indicating that the overexpression of MdRGL2acauses a reduction in hypocotyl response to exogenous GA3.Together, these results indicate significant conservation offunction between MdRGL2a and DELLAs from Arabidopsis.

Discussion

We have identified MdRGL1a/b, MdRGL2a/b, andMdRGL3a/b, apple orthologues of DELLA-encoding genesand members of the plant-specific GRAS family. Like other

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Fig. 2 Unrooted neighbor-joining distance tree of DELLA proteinsfrom apple and other species. Numbers at nodes indicate bootstrapvalues (500 replicates) and the horizontal bar represents a distance of0.1 substitutions per site. The GenBank accession numbers are:DaGAIA (AF492562) (Dubautia arborea), LeGAI (AY269087)(Lycopersicum esculentum), MdRGL2a (DQ007883), MdRGL2b(DQ007884) (Malus domestica), RGL1 (AY048749), RGL2

(AAF01590), RGL3 (AJ224957) (Arabidopsis thaliana), VvGAI1(AF378125) (Vitus vinifera), GAI (Y11337) (Arabidopsis thaliana),CmGAIP (AY326306) (Curcurbita maxima), MdRGL1a (DQ007885),MdRGL1b (DQ007886) (Malus domestica), D8 (AJ242530) (Zeamays), SLN1 (AF460219) (Hordeum vulgare), RHT-D1A (AJ242531)(Triticum aestivum), SLR1 (AB030956) (Oryza sativa), MdRGL3a(DQ007887), MdRGL3b (DQ007888) (Malus domestica)

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members of the subfamily, the MdDELLAs all feature twosignature motifs, DELLA and TVHYNP (Lee et al. 2002;Peng et al. 1997, 1999; Silverstone et al. 1998). Thesedomains are implicated in the perception of GA and thesubsequent destabilisation of the DELLA proteins (Dill etal. 2001; Fu et al. 2002; Gubler et al. 2002; Itoh et al. 2002;Silverstone et al. 2001). Mutation to either of these domainshas been shown to result in a semi-dominant,GA-insensitive, dwarfed phenotype in diverse plant species

(Boss and Thomas 2002; Chandler et al. 2002; Hynes et al.2003; Itoh et al. 2002; Peng et al. 1997, 1999).

Like other DELLA proteins, the N-terminal portions ofthe MdDELLAs are relatively divergent outside theDELLA and TVHYNP domains. In contrast, the C-terminaldomains of MdDELLAs are highly homologous and haveseveral conserved motifs that are shared by most GRASproteins (Bolle 2004; Pysh et al. 1999; Tian et al. 2004).The VHIID, PFYRE and SAW motifs are presumed

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Fig. 3 Transcript levels ofMdRGL1, MdRGL2, andMdRGL3 in different tissues anddevelopmental stages. ThemRNAs were quantified by real-time PCR as described in theMaterial and methods section,and the MdDELLA mRNA copynumber was normalised to thatof MdGAPDH. RNA was iso-lated from apices of spur-typeshoots that had arrested growthand initiated budscales, apicesof extension shoots still initiat-ing vegetative leaves, andexpanding leaves collected inearly summer, 53 days after fullbloom (DAFB). Vegetative andfloral buds were collected in theautumn after fruit harvest, butbefore true dormancy (193DAFB). Germinating seeds werevernalised for 8 weeks at 4°C,then imbided 3 days (3d) and8 days (8d). The means of threeexperiments ±SE are shown

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Fig. 4 Expression of p35S:MdRGL2a in Arabidopsis causes adwarfed phenotype. a Real-time(PCR) was used to quantifyMdRGL2a transgene expression infive independent lines (1, 2, 4, 5, 6)and empty vector control plants(C). MdRGL2a expression isnormalised against that of Atactin.p35S:MdRGL2a transgenic plantswere compared to controls in termsof b stem height along primaryinflorescence axis. c Diameter ofrosette leaves, and d time toflowering in short days (SD, 8 hlight/16 h dark). For b and c, 10–20 individuals per line were grownin long days (16 h light/8 h dark)for 25 days then analyzed. Becausethe majority of p35S:MdRGL2aplants had not flowered by140 days in SD conditions, the datain d is represented as percentageflowering by 94 days in SD. Inb, c, vertical bars represent ±SE

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repression domains and the leucine heptad repeats (LHR)are essential for dimer formation (Itoh et al. 2002). Likeother DELLA proteins, MdDELLAs have a bipartitenuclear localisation signal (NLS), which is consistent withtheir presumed role as transcriptional regulators (Tian et al.2004). The LXXLL motif, which appears in the DELLAsubfamily and in other GRAS proteins, has been shown tofacilitate the interaction of proteins with nuclear receptorsin mammals (Heery et al. 1997).

The six MdDELLAs reported in this paper were identifiedfrom EST libraries, which may not contain genes expressed atvery low levels. Low stringency Southern blot and degeneratePCR analysis failed to identify additional DELLA-encodinggenes in the apple genome (data not shown). Our dataindicate that we have identified all six DELLA-encodinggenes in apple. Arabidopsis has five members of the DELLAsubfamily, whereas monocots such as rice and barley onlyhave one (Chandler et al. 2002; Lee et al. 2002; Ogawa et al.2000). GAI/RGA and RGL2/RGL3 map to duplicated seg-ments of the genome, and have evolved partially redundantfunctions (Dill and Sun 2001; King et al. 2001). Analysis ofmultiple loss-of-function mutants in a GA-deficient back-ground indicates that GAI/RGA are the major repressors ofvegetative and inflorescence growth, RGA/RGL1/RGL2regulate GA-induced floral development, and RGL1/RGL2act in seed germination (Cheng et al. 2004; Lee et al. 2002;Tyler et al. 2004).

Recent molecular studies suggest that Maloideae hasundergone genome hybridisation, with species of theSpiraeoideae subfamily being the most probable parentallineages (Evans and Campbell 2002). Therefore, it is likelythat the a/b pairs of MdDELLAs represent homologousgenes with similar or overlapping functions. We aregenerating single and multiple gene knockouts of theMdDELLAs to determine their role in apple growth anddevelopment. Until these trees are mature (3–5 years), wemust rely on expression data and overexpression pheno-types in heterologous systems to suggest gene function. Thenames of the individual MdDELLAs introduced here arenot intended to imply functional homology to specificArabidopsis RGL proteins.

Expression of MdRGL2a in Arabidopsis causes areduction in leaf size and stem height, and delays floweringin short days. Compared to controls, p35S:MdRGL2aseedlings have shorter hypocotyls and exhibit a reducedelongation response to exogenous GA. These phenotypesrange in severity and are generally correlated with thelevels of MdRGL2a expression. Overexpression of DELLAproteins in Arabidopsis, rice and tobacco have been shownto cause a range of dwarfed phenotypes that vary accordingto the transgene expression level (Ait-ali et al. 2003; Fleckand Harberd 2002; Fu et al. 2001; Hynes et al. 2003; Itoh etal. 2002). The p35S:MdRGL2a dwarfed phenotypes suggestthat MdRGL2a gene function as a negative regulator ofGA-dependent processes is at least partially conservedbetween apple and Arabidopsis.

Recent experiments in Arabidopsis indicate that RGAand GAI respond to auxin and ethylene as well as GAsignals (Achard et al. 2003; Fu and Harberd 2003). Theseobservations support a model in which DELLA proteins arecentral control points that integrate information from anumber of signalling pathways and repress growth untilconditions are optimal (Achard et al. 2006; Alvey andHarberd 2005). In apple, the termination of vegetativegrowth and transition to floral commitment is a complexprocess that appears to be regulated by light intensity,temperature, vernalisation and non-autonomous rootstockeffects (Hirst and Feree 1995; Johnson and Lakso 1985).Spur-type shoots terminate growth in early summer,whereas extension shoots continue leaf initiation andinternode elongation for 4–8 weeks (Fulford 1965, 1966).It has been demonstrated that the endogenous levels ofhighly bioactive polar GAs are significantly lower in theapices of spur-type apple shoots than in the apices ofextending shoots (Looney et al. 1988). Expanding leavesare known to contain high levels of GAs (Aharoni andRichmond 1978; Choi et al. 1995; Jones and Phillips 1966).Given the inverse relationship between localised levels ofbioactive GAs and DELLA activity, it is perhaps notsurprising that we found that all the MdDELLAs, and

0

1

2

3

4

5

6

MSMS+GA3

C 2 1

Hyp

ocot

yl le

ngth

(m

m)

5Fig. 5 The effect of exogenous GA3 on hypocotyl length in threelines of Arabidopsis expressing p35S:MdRGL2a (1, 2, 5) and emptyvector control (C). Seeds were germinated on 0.5X MS in the presence(+GA3, red) or absence (blue) of 10

−4 M GA3 and grown in light (16 hlight/8 h dark) for 7 days. Mean hypocotyl lengths (N=25) are shownwith SE as vertical bars. One set of data from three replicatedexperiments is shown

194 Tree Genet Genomes (2007) 3:187–197

especially MdRGL3, are expressed at much higher levels inrecently arrested spur shoot tips than in growing shoot tipsor expanding leaves. These findings are consistent with thecurrent understanding of DELLA proteins as GA-respon-sive repressors of plant growth.

We observed that MdRGL1 and MdRGL2 are expressedat higher levels in autumn vegetative buds than in recentlyarrested spur buds. While both bud types had a vegetativeterminal meristem at the time of sampling, there are majordifferences in meristem identity between these two samples.Previous studies on Royal Gala have established that thevast majority of spur meristems undergo transition to floralcommitment in early summer, whereas the autumn budshave passed competency to become floral and will arrest asvegetative meristems (Foster et al. 2003). Based on theseobservations and the expression profiles of the MdDELLAs,we speculate thatMdRGL3 holds the meristem in a temporaryarrested state until floral commitment, whereas MdRGL1 andMdRGL2 restrict growth of the vegetative meristem in a long-term capacity. Gene knockouts of individual MdDELLAs willbe essential to test this hypothesis.

Our data indicate that all six MdDELLAs are upregulatedin autumn vegetative buds relative to floral buds. Applefloral buds terminate in a determinate inflorescence andnew growth is initiated from one or more axillary meristem(s), which are repressed until spring. For this reason, wehypothesise that floral buds may require lower levels ofMdDELLA expression to maintain dormancy. Anotherdevelopment is a GA-mediated process that occurs in floralbuds before dormancy, consistent with the lower MdDELLAexpression observed in these tissues.

In Arabidopsis, GA promotes flowering by targetingDELLA proteins for destruction, which ultimately leads toactivation of LEAFY, a key floral meristem identity gene(Achard et al. 2004; Blazquez et al. 1998). In contrast,specific GA species repress flowering in many woodyperennials such as grape and apple (Greene 1989). GA4+7 isroutinely applied to apple buds soon after full bloom toreduce flowering in the current season, thereby increasingflowering in the following year (Greene 1989). Ourobservation of increased MdDELLA expression in meri-stems undergoing transition to floral commitment isconsistent with MdDELLAs being positive regulators offlowering. We are testing this model by overexpressingeach of the MdDELLAs in apple. Expression of MdRGL2ain Arabidopsis significantly delays flowering in short days.However, this likely reflects the ability of MdRGL2a tofunction analogously to the endogenous DELLA proteins,which repress flowering in Arabidopsis.

Certain GA responses such as internode elongation, leafexpansion, and seed germination are conserved betweenspecies, whereas the relationship between GA and flower-ing is not. Changes in the relationships between key

regulatory proteins can lead to rapid morphological evolu-tion (Doebley and Lukens 1998; Purugganan 2000).Mutations to non-coding regions of DELLA genes areassociated with the evolution of novel growth habits in theHawaiian Silversword and altered flowering time in maize(Remington and Purugganan 2002; Thornsberry et al.2001). To better understand the role of MdDELLAs inflowering, we are examining the relationship between theMdDELLAs and the apple LFY orthologues, AFL1 andAFL2 (Wada et al. 2002).

All of theMdDELLAs are expressed at relatively low levelsin germinating apple seeds. In Arabidopsis, RGL1, RGL2 andRGL3 are expressed at high levels only in germinating seedsand/or in flowers and siliques. (Lee et al. 2002; Tyler et al.2004). Based on genetic data and gene expression patterns, itis thought that RGL1, RGL2, and RGL3 inhibit elongation ofthe root pole until conditions are met for germination. UnlikeArabidopsis seeds, apple seeds require several weeks of lowtemperatures (0–4°C) to germinate (Thévenot and Côme1983). Apple seed coats are unusually rich in phenoliccompounds, which trap oxygen and inhibit embryo growth(Côme and Thévenot 1982). Thus, the germination of appleseeds may be more regulated by environmental and physio-logical factors than by the activity of the MdDELLAs.

SLR (rice) and SLN (barley) mRNAs are preferentiallyexpressed in certain tissues (Chandler et al. 2002; Ogawa etal. 2000). In contrast, RGA and, to a lesser extent, GAI areexpressed constitutively in all tissues and appear to beregulated primarily by proteolysis (Fu et al. 2004; Lee et al.2002; Silverstone et al. 1998; Tyler et al. 2004). OnlyRGL1, RGL2 and RGL3 appear to be regulated at thetranscript level (Lee et al. 2002; Tyler et al. 2004; Wen andChang 2002). All of the MdDELLAs appear to betranscriptionally regulated, although protein activity is alsoexpected to be regulated by post-translational modificationsand proteolysis. We are developing antibodies to individualMdDELLAs to quantify protein levels and determineexpression patterns during development. Gene knockoutsof individual MdDELLA genes and further characterisationof the MdDELLA proteins using antibodies will helpelucidate the role of these proteins in apple development.

Acknowledgments The authors thank Qing Deng and KarrynGrafton for excellent technical assistance, Duncan Stanley forphylogenetic advice, and Kimberley Snowden and Robyn Johnstonfor helpful comments on the manuscript. This research was funded bythe New Zealand Foundation for Research Science and Technology.

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