Plant Molecular Biology 46: 373–382, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Up-regulation of genes encoding novel extracellular proteins during fruitset in pea

Manuel Rodrıguez-Concepcion1, Alicia Perez-Garcıa and Jose Pıo Beltran∗Instituto de Biologıa Molecular y Celular de Plantas, U.P.V.-C.S.I.C., Camino Vera s/n, 46022 Valencia, Spain(∗author for correspondence; e-mail jbeltran@ibmcp.upv.es); 1present address: Departament de Bioquımica iBiologia Molecular, Universitat de Barcelona, Martı i Franques 1–7, 08028 Barcelona, Spain

Received 12 September 2000; accepted in revised form 6 February 2000

Key words: differential expression, extracellular proteins, fruit development, gibberellins, Pisum

Abstract

The transition from the carpel of the flower to a developing fruit is a poorly characterized process despite itsagricultural importance. We have identified two genes, GIC19 and GIC4, which are expressed after induction ofpea (Pisum sativum L.) fruit set either by exogenous gibberellins or by pollination. GIC19 expression is temporallyand spatially regulated, with transcripts mainly found in growing carpels and young fruit. Similar to GIC19, GIC4expression is developmentally regulated during carpel and fruit development. However, GIC4 transcripts are foundin other growing tissues throughout the plant. Analysis of their sequences and localization of fusion proteins withGFP indicate that both GIC19 and GIC4 are extracellular proteins. While GIC19 is a small proline-rich proteinwith no overall homology to other reported proteins, GIC4 belongs to a novel family of proteins. Our resultsreinforce a model of gibberellin mode of action during pea fruit set and development involving enhanced synthesisof extracellular proteins and secretory activity to provide materials and energy for cell growth.

Introduction

The molecular mechanisms that control the transfor-mation of carpels into fruit (fruit set) and the eventsthat result in fruit growth and development are poorlyunderstood although these processes are relevant froman agricultural point of view. Plant hormones suchas gibberellins (GAs), auxins and cytokinins have akey role during fruit set (Gillaspy et al., 1993). Inpea (Pisum sativum L.), GAs produced after fertil-ization of the ovules are the key regulatory signalfor induction of fruit set and development (García-Martínez et al., 1991; Santes and García-Martínez,1995; Rodrigo et al., 1997). Self-pollination takesplace one day before anthesis (day −1), and by an-thesis (day 0) fertilization of the ovules has occurred.Removing the anthers from the flower two days beforeanthesis (day −2) prevents pollination and subsequentfertilization. The resulting unpollinated carpels keepgrowing up to day +2 (Figure 1). In the absence of astimulus for fruit set, however, both growth and assim-

ilate import by the carpel cease and eventually carpelslose weight and degenerate (Jahnke et al., 1989). Be-fore they enter senescence, unpollinated carpels canbe stimulated to develop by exogenous application ofgibberellic acid (GA3; Figure 1), giving rise to seed-less parthenocarpic fruits with the same morphologyas those arisen from fertilization (García-Martínez andCarbonell, 1980; Vercher et al., 1987; Vercher andCarbonell, 1991). GA3-induced parthenocarpic fruitset and development is a valuable experimental systemto study the early changes that take place when thecarpel of the flower is transformed into a developingfruit, since the time of induction can be controlled. Us-ing this system it has been shown that GA3 treatmentrapidly stimulates secretion of acid invertase to theextracellular space (Estruch and Beltrán, 1991) and ac-tivates glucose uptake and metabolism (Beltrán et al.,1991), increasing the sink strength of carpel cells.Eventually, both fertilization and treatment with GA3transform the carpel in a strong sink organ in whichthe activated metabolism supplies energy for cell di-

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Figure 1. Fresh weight of unpollinated carpels from emasculatedflowers either untreated (black circles) or treated with GA3 at day+2 (white circles). Data represent the mean of at least five samples± SE.

vision and expansion processes that result in fruitgrowth (Ho, 1988; Jahnke et al., 1989; Beltrán et al.,1991). To characterize genes that participate in fruitset and development, we have searched a cDNA li-brary for differentially expressed genes in GA3-treatedpea carpels. In this paper we report the identificationof two genes (GIC19 and GIC4) whose expressionis up-regulated upon induction of fruit set by eithertreatment of carpels with GA3 or fertilization, andshow that the corresponding proteins are targeted tothe extracellular space, where they likely have a rolein supporting the active cell growth that takes placeafter stimulation of fruit set and development.

Materials and methods

Plant material and treatments

Pea (Pisum sativum L. cv. Alaska) plants weregrown as described (Rodríguez-Concepción and Bel-trán, 1995). Unpollinated pea carpels were obtainedby removing petals and stamens from flowers twodays before anthesis (day −2). At the beginning ofday +2, fruit set was induced by applying directly tothe carpels of emasculated flowers 20 µl of 0.3 mMgibberellic acid (GA3, Fluka) in 0.1% v/v Tween 80.Control and treated samples were harvested at thesame time, weighed, frozen in liquid nitrogen, andstored at −70 ◦C until used for RNA blot experiments.Pollinated fruit were collected from intact flowers leftto self-pollinate.

Differential screening

A cDNA library from pea carpels treated with GA3was constructed in λNM1149 vector as described(Sommer et al., 1990). Clones corresponding to differ-entially expressed mRNAs were identified by screen-ing the library with single-strand cDNA probes syn-thesized from poly(A)+ mRNA isolated from carpelseither treated for 9 h with GA3 (probes +) or untreated(probes −), as described (Rodríguez-Concepción andBeltrán, 1995). The cDNA inserts of differentialclones were rescued by PCR using primers from bothsides of the phage EcoRI cloning site. PCR prod-ucts were subcloned in pT7Blue (Novagen) for furthermanipulations.

Nucleic acid analysis

The cDNA inserts cloned in pT7Blue were sequencedusing vector primers and specific primers. Sequenceanalyses were performed using the GCG and MacVec-tor 3.5 packages. Poly(A)+ mRNA was isolated fromfrozen tissue using the Dynabeads oligo (dT)25 pro-cedure (Dynal) and eluted in 2 mM EDTA, 50% v/vformamide. For RNA blot analysis, 2 µg of everypoly(A)+ mRNA sample was fractionated on 1.2%w/v agarose gels containing 7% v/v formaldehyde andtransferred onto Hybond N+ nylon filters (Amersham)by capillary transfer. A RNA size marker (0.24–9.5 kb RNA ladder, Gibco-BRL) was used to estimatethe length of the mRNAs which hybridized with theprobes. Equal loading of mRNA was verified by usinga GAPDH constitutive probe (Rodríguez-Concepciónand Beltrán, 1995). Probes were made from the entirecloned cDNA sequences 32P-labelled by the randomhexamer priming method following the protocol sup-plied by Amersham. Hybridization was performedovernight at 65 ◦C in 0.3 M sodium phosphate pH 7.2,7% w/v SDS, 1 mM EDTA. Washes were carried outtwice in 2× SSC/0.1% w/v SDS at room temperature,and twice in 0.1× SSC/0.1% w/v SDS, 15 min each,at 65 ◦C. Filters were exposed either to X-ray filmat −70 ◦C with intensifying screens, or to BAS MP2040S imaging plates for the BioImaging AnalyzerBAS 1500 (Fujifilm).

Tissue print RNA blot

Pea seedlings were obtained from seeds germinatedon filter paper moistened with distilled water in petridishes and grown at room temperature for five days.Equidistant marks were then drawn on the epicotyl

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Figure 2. Analysis of GIC19 and GIC4 expression in pea carpels.Poly(A)+ mRNA (2 µg) was isolated from the following samples:unpollinated carpels at day 0, unpollinated carpels at day +2 eithertreated (+) or not (−) with GA3 for the indicated times (hours),and pollinated carpels at day +2 (p+2). RNA samples were elec-trophoresed and transferred onto a membrane. The same blot washybridized with cDNA probes GIC19, GIC4, and a constitutive (C)GAPDH probe to monitor loading. The estimated size (kb) of themRNAs hybridizing with the probes is indicated.

stem with a permanent marker. Growth was inducedwith GA3 applied to the apical region of the epicotylas described above for carpels. After 25 h, seedlingswere photographed to record the increase in stemlength. Tissue print RNA blots were made as described(Ye and Varner, 1991) with longitudinal sections ofthe stem made with a sharp razor blade and pressedagainst a Hybond N+ nylon membrane (Amersham).

Particle bombardment

The pT7Blue plasmids with the GIC19 and GIC4cDNA inserts were used as templates for PCR re-actions to incorporate restriction sites in order toclone the corresponding cDNA sequences in framewith the GFP first ATG codon in the pGFP-MRCvector (Rodríguez-Concepción et al., 1999). Spe-cific primers were used for GIC19 (G19F, 5′-GGTACCCAATGGTTTTAATTTCTTTTACTTGTTTCTGC-3′; G19R, 5′-GTCGACAATGCATTGTACTCATAGGTAC-3′) and GIC4 (G4F, 5′-CTCGAGGTACCGTGAATGTTCATTGAC-3′; G4R, 5′-CTACGCCATGGACGACAAGATCAATGA-3′). PCR products werecloned in pCR2.1-TOPO (Invitrogen) to create pCR-G19 and pCR-G4, and sequenced to confirm thatthey corresponded to the expected sequences. TheGIC19 PCR sequence was released from the pCR-G19 plasmid with EcoRI and cloned into pGFP-MRCin frame with the amino terminus of GFP to expressthe chimeric GIC19-GFP protein under the control ofthe 35S promoter in plant cells. A similar approachcreated GIC4-GFP by cloning the XhoI/NcoI GIC4

PCR insert from pCR-G4 into the same sites of pGFP-MRC. Transient expression of the GFP-fusion proteinsin onion bulb epidermis cells was carried out by mi-crobombardment with M17 tungsten particles coatedwith the corresponding pGFP plasmid DNA by using aBiorad Biolistic PDS-1000/He system at a pressure of7.6 MPa as recommended by the manufacturer. Greenfluorescence corresponding to the GFP fusion proteinswas examined directly with a Nikon epifluorescencemicroscope.

Results

Induction of pea fruit set correlates with GIC19 andGIC4 expression

Controlled induction of fruit set and development inpea was accomplished by applying GA3 to unpolli-nated ovaries (from emasculated flowers) at day +2(Figure 1). To identify genes differentially expressedafter induction, we screened a cDNA library fromGA3-treated unpollinated carpels using probes + (syn-thesized from mRNA isolated from carpels treatedwith the hormone) and − (from untreated ones). Weanalysed 100 000 plaques and isolated two cDNAclones (GIC19 and GIC4, for gibberellin-inducedcarpel genes) which showed a stronger hybridizationsignal with the probes +. Inserts were rescued by PCRand radioactive probes were made to study the expres-sion patterns of the corresponding genes during thetransition from carpel to developing fruit (Figure 2).

The size of GIC19 and GIC4 transcripts was es-timated to be ca. 0.3 kb and 0.8 kb, respectively, bycomparison with size standards in RNA blots. GIC19mRNA levels decreased in carpels from day 0 upto the beginning of day +2. When carpels stoppedgrowing later during day +2 (Figure 1), transcriptsbecame undetectable (Figure 2). Fruit set inductionby GA3treatment resulted in an increased level ofGIC19 RNA. GIC19 transcript level in pollinatedfruit, however, was much higher than in GA3-inducedparthenocarpic fruit (Figure 2). Similar to GIC19,GIC4 gene expression was already found in growingcarpels at the day of anthesis. At day +2, however,unstimulated carpels showed no detectable GIC4 tran-scripts (Figure 2). Stimulation of fruit set with GA3(Figure 1) correlated with induction of GIC4 gene ex-pression 10 h after treatment. Unlike GIC19, GIC4transcript level in young fruit induced to set by fertil-ization was very similar to that found in GA3-induced

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Figure 3. RNA blot analysis of GIC19 and GIC4 expression in pea organs. Samples of 2 µg of poly(A)+ mRNA from different plant tissueswere used in RNA blot experiments with probes made from the GIC19 and GIC4 cDNAs. A constitutive (C) GAPDH probe was used tomonitor the amount of RNA loaded in each lane. A. Expression in flower organs at day −2. Se, sepals; Pe, petals; St, stamens; Ca, carpels. B.Expression in pea plant organs: YL, young apical leaves; L, mature expanded leaves; F, flowers at day 0; S, stems; R, roots.

parthenocarpic fruit (Figure 2). Altogether, we haveisolated two cDNAs (GIC19 and GIC4) correspond-ing to developmentally regulated genes with differentexpression patterns. However, both of them were ex-pressed in growing carpels and repressed when growthstopped. Also similarly, their mRNAs were detectedwhen fruit set and development was induced by eitherGA3 treatment or pollination.

GIC19 is mainly expressed in the carpel but GIC4expression correlates with cell growth throughout theplant

To study whether the GIC genes had a role in otherdevelopment processes in the pea plant we investi-gated their patterns of expression in different tissuesby RNA blot analysis. Figure 3A shows that in pre-anthesis flowers (day −2), the bulk of GIC19 mRNAwas found in carpels. Much lower levels were foundin petals, and no expression was detected in sepalsand stamens (Figure 3A). Other plant organs were alsotested for expression of GIC19 (Figure 3B), showingthat GIC19 was predominantly expressed in flowers,although low levels of cross-hybridizing transcriptswere also detected in roots (Figure 3B).

In flowers at day −2, GIC4 transcripts were notdetected in sepals but were present in the other threewhorl organs (Figure 3A). The lowest transcript levelwas found in carpels and the highest in petals. Atthis stage of development, sepals are already formed,whereas stamens and carpels are still growing andpetals are in a very active cell expansion process thatwill lead to full blossom at anthesis. Therefore, GIC4expression correlated well with cell growth activity inflower organs. In vegetative tissues, GIC4 mRNA wasfound in all the organs analysed, although they were

Figure 4. GIC4 expression in the pea epicotyl. Marks were drawn0.5 cm apart on the stem of a 5-day old pea seedling (A).Twenty-five hours after applying GA3 to the apical region of theseedling, the separation of the marks indicated the elongation of thestem (B). The epicotyl from the GA3-treated seedling shown in Bwas used for RNA tissue printing and the blot hybridized with aGIC4 probe (C).

almost undetectable in roots (Figure 3B). Transcriptlevels in apical young growing leaves were higher thanthat in mature expanded leaves, again showing a posi-tive correlation between GIC4 transcript accumulationand cell growth. The amount of GIC4 transcripts ob-served in stems was highest in vegetative tissues andit was similar to the level observed in flowers (Fig-ure 3B). To test whether GIC4 gene expression alsocorrelated with stem growth, GIC4 transcript levelswere monitored in the apical region of pea epicotylsinduced to grow with GA3. RNA tissue-printing ex-periments clearly showed the accumulation of GIC4transcripts in the stem elongation area (Figure 4), sup-porting the proposed role for GIC4 in cell growthprocesses.

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Figure 5. GIC19 cDNA sequence, deduced ORF, and hydrophilicity plot. A polyadenylation signal is underlined on the GIC19 cDNA sequence(accession number Z67874). The star indicates the predicted signal peptide cleavage site. PAAA motifs are marked in bold and (P)2−3 repeatsare underlined on the amino acid sequence. The hydrophilicity plot of the deduced amino acid sequence was constructed over a window ofseven residues using MacVector 3.5. Positive values indicate hydrophilic regions and negative values represent hydrophobic regions. An arrowindicates the predicted signal peptide cleavage site.

GIC19 is a proline-rich protein

To address the putative function of the GIC genes,the cDNA clones were sequenced and analysed. Fig-ure 5 shows the sequence of the cloned GIC19 cDNA.The cDNA is 351 bp in length and the first ATGcodon is found at position 20 in the cDNA. Al-though a reading frame with no stop precedes thisATG, 5′-RACE (rapid amplification of cDNA ends)experiments with RNA from pea carpels of severaldevelopmental stages could not detect a longer 5′ re-gion, suggesting that the ATG at position 20 is thetranslation start point and that the reported cDNA is,at least, nearly full-length. Consistently, the length ofthe cDNA shown in Figure 5 is in agreement withthe size of the corresponding GIC19 transcripts ob-served in RNA blots (Figure 2). The protein sequencededuced from this cDNA shows a predicted highly hy-drophobic N-terminal region (Figure 5B). In addition,residues A at position 17 and V at position 15 fit the(−1, −3) rule used to predict signal peptide sequenceprocessing sites (von Heijne, 1986). Assuming thatthis N-terminal region may constitute a signal peptide,the putative mature GIC19 protein would be a smallprotein of 75 amino acid residues (8180 Da) rich inP (13.33%), A (12.00%) and C (10.67%) residues,and with a predicted isoelectric point of 9.12. The P

and A residues are usually organized in repeats: twoPAAA and three PP (Figure 5). Although GIC19 doesnot show significant homology with any other proteinin the databases, some features of its sequence suchas proline-richness, repeated motifs, basic nature andpresence of a putative signal peptide sequence are gen-erally found in cell wall proteins (Cassab and Varner,1988; Showalter, 1993; Cassab, 1998).

GIC4 belongs to a family of acidic proteins

The cloned GIC4 cDNA is 770 bp long (in agreementwith the size of the corresponding GIC4 mRNA; Fig-ure 2), and the first ATG codon is found at position 130(Figure 6A). The nucleotide sequence flanking thisATG codon (AAAATGGC) is in close agreement withthat described as a consensus in plant genes for theinitiation of protein synthesis (ACAATGGC) (Lütkeet al., 1987), strongly suggesting that this is indeed thetranslation initiation point. The following open read-ing frame is interrupted at position 613 by a TGA stopcodon, and the resulting polypeptide has 161 aminoacid residues, a molecular mass of 17 507 Da and anestimated isoelectric point of 3.96. A potential glyco-sylation motif (NET; Kornfield and Kornfield, 1985)is present at position 14 of the protein (Figure 6A).

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Figure 6. GIC4 sequence analysis. A. GIC4 cDNA sequence (accession number Z47790), deduced amino acid sequence, and hydrophilicityplot. A polyadenylation signal is underlined on the nucleotide sequence and the VXEXA repeats are underlined on the protein sequence. B.Alignment of pea (Pisum) GIC4 amino acid sequence with similar proteins from chickpea (Cicer), potato (Solanum), Arabidopsis, buckwheat(Foagopyrum), Alnus, rubber tree (Hevea) and kiwi (Actinia). Residues identical to those in the GIC4 sequence are boxed in black, andother conserved residues are boxed in grey. Numbering indicates amino acid position. Gaps were introduced by the CLUSTALW program tomaximize similarity.

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The predicted GIC4 protein is rich in glutamic acid (E,22.98%), valine (V, 14.29%), and alanine (A, 13.66%).

Several proteins similar to GIC4 were found in theNCBI database. These proteins appear to have rolesin very different processes, since they are encoded bygenes induced during infection in Alnus glutinosa rootnodules (AG13, Guan et al., 1997; 33.1% similaritywith GIC4), fruit development of buckwheat (acces-sion number 2317676; 33.1%) and kiwi (KIWI501,Ledger and Gardner, 1994; 33.7%), osmotic stress inetiolated chickpea epicotyls (2961298; 32.4%), andearly tuberization in potato (TUB8, Taylor et al., 1992;34.8%). The highest similarity was found with an al-lergen from rubber (HevB5, Slater et al., 1996; 35.8%)and with a polypeptide sequence deduced from anArabidopsis EST (3157925; 41.4%) (Figure 6B). Theexpression of KIWI501 was detected in young fruitafter fruit set, similarly to that described for GIC4(Figure 2). Although more information about the ex-pression patterns and regulation of the correspondinggenes is necessary, there are some common featuresthat suggest that these proteins may belong to a familyof acidic proteins with similar functions. For instance,the sequence following the first methionine residue ishighly conserved: MA(T/S/A)VEV (Figure 6B). Be-sides that, the similarity among these proteins wasmainly due to the abundance of E and A residues, usu-ally grouped in short repeats. In GIC4, besides 7 EE(most of them followed directly by an A residue) and3 AA repeats, 5 pairs of T residues either followedor preceded by E residues were found (Figure 6B).Longer repeats were also distributed throughout thesequence of these proteins. For instance, repeats ofthe pentapeptide VXEXA, in which X was usuallyE or A, were found four times in GIC4 and werepresent in other proteins of the family (Figure 6B). Therepeats and the proline-richness of most of these pro-teins suggests that they might be structural acidic cellwall proteins, although they do not show homologyto any of the well-defined plant proline-rich proteinsand the repetitive motifs are not characteristic (Cassaband Varner, 1988; Showalter, 1993; Cassab, 1998).Cell wall localization has been proposed for TUB8and AG13 (Taylor et al., 1992; Guan et al., 1997),but no experimental evidence is available yet. In thecase of AG13, the authors report that the N-terminusof the protein could serve as a signal peptide sequence(Guan et al., 1997). In GIC4, however, the N-terminalregion is not particularly hydrophobic (Figure 6A). Inaddition, an important difference between GIC4 andthe rest of the proteins of the family is that GIC4 is not

Figure 7. Subcellular localization of GFP-tagged GIC19 and GIC4in plant cells. Onion bulb epidermis layers were bombarded withparticles coated with constructs to express GFP (A), GIC4-GFP(B), or GIC19-GFP (C). Green fluorescence corresponding to thelocalization of the proteins was detected by epifluorescence.

rich in proline. Therefore, experiments to determinethe subcellular localization of this protein are essentialto ascertain whether or not GIC4 is a cell wall protein.

GFP-labelled GIC19 and GIC4 localize to theextracellular space of plant cells

The features of GIC19 amino acid sequence sug-gest that it may be a component of the cell walland/or the extracellular matrix of carpel cells. Onthe other hand, although GIC4 is not proline-richand lacks a clear N-terminal signal peptide sequence,the similarity with the rest of the homologous pro-teins and the presence of repeated regions suggestthat it might also be an extracellular protein. To de-termine the subcellular localization of these proteinsin vivo, we fused them to the N-terminus of sGFP,a soluble codon-optimized green fluorescent protein(Rodríguez-Concepción et al., 1999). The fusion pro-

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teins were transiently expressed under the control ofthe 35S promoter in onion epidermis cells by par-ticle bombardment (Figure 7). Green fluorescencecorresponding to GFP is localized to the nucleus andthe cytoplasm of onion cells. The fusion proteinsGIC19-GFP and GIC4-GFP, however, were distrib-uted throughout the periphery of bombarded cells(Figure 7), consistent with their proposed localizationin the cell wall. Some fluorescence appeared to dif-fuse to the periphery of neighbouring cells, suggestingthat the fusion proteins were indeed secreted and thendistributed in the apoplastic space of cell walls. There-fore, our results suggest that GIC19 and GIC4 areproteins with a role in the cell wall or the extracellularmatrix of plant cells.

Discussion

Here we report the identification of GIC19 and GIC4,two pea genes which are induced when the carpelis transformed into a developing fruit. The low levelof GIC19 transcripts in pea organs other than youngcarpels and fruit (Figure 3), and the lack of expressionin non-growing carpels (Figure 2), support the notionthat its main role is associated with carpel and fruitgrowth and development. The fact that GIC19 tran-script levels in pollinated fruit are much higher thanin GA3-induced parthenocarpic fruit (Figure 2) sug-gests that other factors besides GAs which regulatepea fruit set (Gillaspy et al., 1993) may also regulatethe expression of GIC19 in fruit induced to set anddevelop by fertilization. Consistently, auxins and cy-tokinins also induce GIC19 expression in pea carpels(Rodríguez-Concepción and Beltrán, unpublished re-sults). Although GIC19 shows no overall homologyto any other protein, it shares similar sequence fea-tures with other plant proline-rich proteins (PRPs).PRPs are a family of cell wall proteins proposed tofunction both in maintaining structural integrity ofmature tissues and in determining cell type-specificwall structure during plant growth and development(Showalter, 1993; Cassab, 1998). GIC19 richness inP and A residues organized in repeats was also foundin the C-terminus of the Brassica napus microsporeI3 protein (Roberts et al., 1991), and abundance ofA residues has been also reported in maize antherMSF14 (Wright et al., 1993). Both MSF14 and I3are flower PRPs which are down-regulated in the laterstages of flower development, similarly to GIC19. Al-though they have also been proposed to be associated

with the cell wall, their function remains obscure.The N-terminal domain of GIC19 contains all the Presidues and repeats and the C-terminal domain is richin C residues, a structural organization similar to thatfound in several PRPs including flower proteins suchas tomato TPRP-F1 (Salts et al., 1993), which is ex-pressed in young fruit like GIC19. The interaction ofsome of the proteins of this group with other com-ponents of the extracellular matrix has been found tobe critical for proper developmental processes (Ertlet al., 1992; Cheung et al., 1995). The C-rich andP-rich domains might be involved in the formationof disulfide bonds between these proteins themselvesand/or with other proteins within the extracellular ma-trix. Another characteristic feature of GIC19 proteinis the presence of a palindromic peptide sequence(APPCPPA) from position 44 to 50 (Figure 5). Pep-tide palindromes are common in extracellular matrixproteins, and their presence suggests the existenceof a centrosymmetric site which may contribute toself-assembly (Kieliszewski and Lamport, 1994). Inaddition, the predicted pI of GIC19 is 9.12, suggestingthat this protein may interact with the acidic pectinnetwork within the cell wall. Together, the pattern ofexpression and sequence features of GIC19 suggestthat potential matrix interactions between GIC19 andother components of the cell wall may be critical forcarpel and early fruit growth and development in pea.

GIC4 is a member of a family of proposed ex-tracellular plant acidic proteins (Figure 6B). Here weshow that a member of the family, GIC4, is actuallylocalized to the extracellular space (Figure 7). Pro-teins from this family have been found in a variety oftissues and developmental stages. Consistently, GIC4is expressed throughout the pea plant (Figure 3). Theexpression pattern of GIC4 suggests that the corre-sponding protein may be required in the extracellularmatrix during stages of rapid growth, in agreementwith a role of GIC4 in the cell wall during expansion ofpea cells. In addition, GIC4 transcript level in youngfruit induced to set by fertilization was very simi-lar to that found in GA3-induced parthenocarpic fruit(Figure 2), suggesting that GAs constitutes a majorregulatory stimulus for GIC4 expression.

After fruit set, the pea pod (pericarp) enters anexponential phase of growth (Figure 1) which resultsin dramatic increases in pod length and width duringearly fruit development (Pate and Flinn, 1977). Therapid and significant changes in cell shape that occurduring this period (Vercher et al., 1987; Vercher andCarbonell, 1991) require the synthesis of new cell wall

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material to support cell expansion. Studies on the ul-trastructure of pod cells showed that one of the firstdetectable effects of fruit set induction with GA3 wasthe reorganization of the membrane systems likely di-rected to the synthesis of primary cell wall (Vercherand Carbonell, 1991). By contrast, in untreated carpelsa typical senescence process is initiated involving dis-organization of cell wall components. The expressionof genes such as GIC19 and GIC4, which encodecell wall or extracellular matrix proteins, is there-fore consistent with the physiology of fruit set. BothGIC genes are expressed in the carpel pericarp (Pérez-García, Rodríguez-Concepción and Beltrán, unpub-lished results), where their expression appears to benecessary for the cells to rapidly grow after stimula-tion by GAs either exogenously applied or producedafter fertilization of the ovules. Genes such as thoserepresented by tomato TPRP-F1 (Salts et al., 1993),which was proposed to be a proline-rich cell wall pro-tein and shares similar features with GIC19, are alsoexpressed during early stages of fruit growth in otherplants. Similarly, treatment of gibberellin-deficientmutants with GA3 induced the accumulation of thesmall cell wall proteins GAST1 (Shi et al., 1992)and At2191 (Phillips and Huttly, 1994) in tomato andArabidopsis flowers, respectively.

Besides an induction of the expression of genesproviding cell wall material, growth stimulated byGAs is also associated with an enhancement of se-cretion mechanisms (Jones and Moll, 1983; Fincher,1989; Estruch and Beltrán, 1991). In pea, one ofthe first visible effects after GA3-induced fruit set isthe formation of cytoplasmic vesicles that fuse withthe plasma membrane in pod cells, likely contribut-ing to release components for cell wall synthesis andincrease plasma membrane surface (Vercher et al.,1987). GA3 application also stimulates trafficking ofacid invertase from the endoplasmic reticulum to theGolgi apparatus on its way to the extracellular space(Estruch and Beltrán, 1991), hence contributing toincrease assimilate unloading and young fruit sinkstrength. Therefore, our results are in agreement withthe current model that the mode of action of GAs dur-ing pea fruit set and development involves stimulationof growth through expression of cell wall proteins andenhanced secretory mechanisms to export these andother proteins (such as invertase) to the extracellularspace. As a result, GAs-activated mechanisms providematerials and energy for cell expansion.

Acknowledgements

We thank Dr N. Campos for critical reading of themanuscript and R. Martínez-Pardo and A. Villar forexcellent care of the plants. This work was supportedby Secretaría General del Plan Nacional de Investi-gación Científica y Desarrollo Contract Grant BIO97-0583 and BIO2000-0940. M.R.C. received pre- andpost-doctoral fellowships from the Spanish M.E.C.

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