1 1 2 3 4 5 6 Running head: Tomato procera - Plant Physiology

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Running head: Tomato procera Mutant and Fruit-Set 7

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Corresponding author: Dr José Luis García-Martínez 9

Instituto de Biologia Molecular y Celular de Plantas (IBMCP) 10

Universidad Politécnica de Valencia-CSIC 11

Ingeniero Fausto Elio s/n 12

46022-Valencia 13

Spain 14

Phone: + 34-963877865; Fax: +34-963877859; e-mail: [email protected] 15

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Research Area: Development and Hormone Action 17

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Plant Physiology Preview. Published on August 31, 2012, as DOI:10.1104/pp.112.204552

Copyright 2012 by the American Society of Plant Biologists

www.plantphysiol.orgon December 26, 2018 - Published by Downloaded from Copyright © 2012 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

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Characterization of the procera Tomato Mutant Shows Novel Functions of 5

SlDELLA Protein in the Control of Flower Morphology, and Cell Division and 6

Expansion and Auxin-Signalling Pathway during Fruit-Set and Development1 7

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Esther Carrera2, Omar Ruiz-Rivero2, Lazaro Eustaquio Pereira Peres, Alejandro 10

Atares, Jose Luis Garcia-Martinez* 11

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Instituto de Biología Molecular y Celular de Plantas (IBMCP), Universidad Politécnica 13

de Valencia (UPV)-Consejo Superior de Investigaciones Científicas (CSIC), Ingeniero 14

Fausto Elio s/n, 46022-Valencia, Spain (E.C., O.R.-R., A.A, and J.L.G.-M.), and 15

Department of Biological Sciences, Escola Superior de Agricultura “Luiz de Queiroz”, 16

Universidade de São Paulo, Av. Pádua Dias, 11, CP 09, Piracicaba, SP 13418-900, 17

Brazil (L.E.P.P.) 18

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Footnotes: 1 1This work was funded by a grant from the Ministerio de Educación y Ciencia of Spain 2

(BIO2009-07968). The work of E Carrera was supported by a Ramón y Cajal grant. 3 2These authors contributed equally to this work. 4

*Corresponding author: e-mail [email protected]; fax + 34-963877859 5

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ABSTRACT 1

procera (pro) is a tall tomato (Solanum lycopersicum L.) mutant carrying a point 2

mutation in the GRAS region of the gene encoding SlDELLA, a repressor in the 3

gibberellin (GA) signaling pathway. Consistent with the SlDELLA loss-of-function, pro 4

plants display a GA-constitutive response phenotype, mimicking WT plants treated with 5

GA3. The ovaries from both non-emasculated and emasculated pro flowers had very 6

strong parthenocarpic capacity, associated with enhanced growth of pre-anthesis ovaries 7

due to more and larger cells. pro parthenocarpy is facultative because seeded fruits were 8

obtained by manual pollination. Most pro pistils had exserted stigmas, thus preventing 9

self-pollination, similar to WT pistils treated with GA3 or auxins. However Style2.1, a 10

gene responsible of long styles in non-cultivated tomato, may not control the enhanced 11

style elongation of pro pistils, because its expression was not higher in pro styles and 12

did not increase upon GA3 application. Interestingly, a high percentage of pro flowers 13

had meristic alterations, with one additional petal, sepal, stamen and carpel at each of 14

the four whorls, respectively, thus unveiling a role of SlDELLA in flower organs 15

development. Microarray analysis showed significant changes in the transcriptome of 16

pre-anthesis pro ovaries compared to WT, indicating that the molecular mechanism 17

underlying the parthenocarpic capacity of pro is complex and that it is mainly 18

associated with changes in the expression of genes involved in GA and auxin pathways. 19

Interestingly, it was found that GA activity modulates the expression of cell division 20

and expansion genes and an auxin signaling gene (SlARF7) during fruit-set. 21

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INTRODUCTION 1

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Fruit-set is thought to be under the control of hormones, mainly auxins and 3

gibberellins (GAs), synthesized in the ovary after pollination/fertilization (Pandolfini et 4

al, 2007; Serrani et al; 2007b, De Jong et al; 2009a, Fuentes and Vivian-Smith, 2009). 5

Auxin controls the synthesis of GA during early fruit development (Ozga and Reinecke, 6

2003; Serrani et al, 2008; Dorcey et al, 2009). In tomato, auxin–induced fruit set is 7

mediated by GA, although auxin may also have a GA-independent effect on fruit 8

growth (Serrani et al, 2008). 9

Parthenocarpic growth can be induced by application of different kinds of 10

hormones (Vivian-Smith and Koltunow, 1999; Serrani et al, 2007a; Dorcey et al, 2009). 11

Indeed, there are mutants with parthenocarpic capacity due to overexpression of genes 12

of auxin biosynthesis (Pandolfini et al, 2007) or the auxin receptor (SlTIR1) (Ren et al, 13

2011), and to down-regulation of transcription factors (TF) involved in the auxin 14

signaling pathway (IAA9 and ARF7) (Wang et al, 2005; Goetz et al, 2007; De Jong et 15

al, 2009b). Parthenocarpic capacity of several tomato mutants (pat, pat-2 and pat-3/pat-16

4) is also associated with higher content of GA and altered expression of GA 17

metabolism genes in the ovary (Fos et al, 2000, 2001; Olimpieri et al, 2007). DELLA 18

proteins, key regulators of the GA-signaling pathway, are destabilized in response to 19

GA through the ubiquitin-26S-proteasome pathway (Sun and Gubler, 2004). Upon GA 20

binding to GID1 (the GA receptor), the GID1-GA complex interacts with the DELLA 21

proteins through the DELLA/TVHYNP motif, allowing its recognition by the F-box 22

protein (SLY1 in Arabidopsis, GID2 in rice) of the Skp1/Culin/F-box (SCF) E3 23

ubiquitin ligase complex, which targets the DELLAs for degradation (Harberd et al, 24

2009; Hirano et al, 2010). Arabidopsis contains five genes encoding DELLA proteins, 25

while rice and tomato, for example, have only one (Martí et al, 2007). The slender la cry 26

DELLA mutant of pea has parthenocarpic capacity (Weston et al, 2008), and antisense-27

SlDELLA plants of tomato have a tall phenotype and produce parthenocarpic (seedless) 28

fruits (Martí et al, 2007). All these results support the pivotal role played by auxin and 29

GA in fruit-set. 30

Diverse mutations in the DELLA/TVHYNP domains transform the DELLA 31

protein into a constitutively active, dominant form, resistant to GA-induced degradation 32

(Harberd et al, 2009). By contrast, null and loss-of-function recessive mutations in the 33

DELLA genes of several species provoke a constitutive GA response phenotype (Sun 34



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and Gubler, 2004; Weston et al, 2008; Harberd et al, 2009). This is also the case of the 1

procera (pro) tomato mutant, caused by a point mutation in the VHIID motif of the 2

SlDELLA gene leading to a single amino acid change (Bassel et al, 2008; Jasinski et al, 3

2008). pro mutants are taller and have non-serrated leaves (Jones, 1987; Jupe et al, 4

1988). Interestingly, their GA content is reduced (Jones, 1987; Van Tuinen et al, 1999), 5

indicating that pro is not a GA metabolic mutation. Remarkably, the dwarf phenotype of 6

the dominant Brassica rapa rga1-d mutant is also produced by the change of one amino 7

acid outside the DELLA domain and within the GRAS region (Muangprom et al, 2005). 8

It has been proposed that, in rice, after DELLA binding to GID1 through the 9

DELLA/TVHYNP motif, the GRAS domain helps to stabilize the complex and to be 10

recognized by the F-box for degradation (Hirano et al, 2010). This hypothesis agrees 11

with the contention that the higher DELLA level and dwarf phenotype of the dominant 12

Brrga1-d mutant is due to reduced interaction of DELLA with the F-box needed for 13

degradation (Muangprom et al, 2005; Hirano et al, 2010). By contrast, in the case of the 14

recessive pro mutant, the mutation may lead to lower activity of the repressor protein, 15

for instance causing reduced or no interaction with downstream target TFs, rather than 16

to reduced F-box binding. 17

In this work we have further characterized the pro mutant, paying particular 18

attention to the reproductive phenotypes that have been overlooked or poorly considered 19

in previous work with the mutant. The pro mutation induces style elongation (thus 20

preventing self-pollination), meristic changes in the flower (increased number of all 21

floral organs), and strong parthenocarpic capacity. Transcriptome analysis and qPCR of 22

selected genes suggest the involvement of specific TFs in the altered morphology of pro 23

flowers. The results unveil new functions for SlDELLA and that GA activity regulates 24

cell division and expansion and the auxin signaling pathway during fruit-set and 25

development. 26

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RESULTS 28

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Phenotypic Characterization of the procera Mutant 30

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Seedlings of the pro mutant had longer hypocotyls and shorter roots, and the 32

plants were slender, with longer internodes and thinner stems than control (Table 1 and 33

Figs 1A and 1B). pro enhanced growth both in plants with low (WT Micro-Tom) and 34



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high (Micro-Tom with introgressed D) brassinosteroid content (Fig. 1C). In pro plants 1

the number of leaves before the first inflorescence was higher (8-9 compared to 6-7 2

leaves in WT) (Table 1), associated with delayed flowering. The leaves had reduced 3

size and in some cases less leaflets, and lacked the characteristic serrated borders of WT 4

(Fig. 1D) (Jasinsky et al, 2008). 5

pro mutant flowers had longer sepals, petals and stamens (Table 1 and Fig. 1E), 6

and their stigma protruded above the staminal cone due to longer style (Table 1 and Fig. 7

1F), also in flowers from D-pro plants (Fig. 1F). Interestingly, the average number of 8

floral organs in all whorls was higher in pro than in WT due to a large number of 9

flowers that beared six sepals, six petals, six stamens and four carpels, compared to five 10

sepals, petals and stamens and three carpels in essentially all WT flowers (Table 1 and 11

Figs 1E and 1G). pro pistils had longer cells, rounded stigma, and the stylar transmitting 12

tissue of those with four carpels was hollow (Figs 1H and 1I), which might affect pollen 13

germination and tube transmission. At late anthesis, when the intersporangial septum of 14

WT anthers had degenerated, it still remained intact in pro anthers, probably due to 15

delayed senescence (Fig. 1H). Cells of style, anther wall, and ovary pericarp of pro 16

flowers at the time of anthesis (d0) were larger than those of WT (Table 1 and Figs 1G 17

and 1I). The number of cell layers in the pericarp was also higher in pro than WT 18

ovaries at anthesis (Table 1). 19

pro had fewer flowers per plant (Table 1), although total number of developed 20

fruits was almost double but smaller (Table 1; see also Figs 2A and 2B). Interestingly, 21

many pro fruits (up to 28% in some batches) grown from spontaneous self-pollinated 22

flowers bore an additional fruit-like structure at the style end (Fig. 2B). Fruit ripening 23

was delayed in the mutant by about 9 d, and the Brix index value of the juice was higher 24

(Table 1). 25

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The procera Mutant Shows Facultative Parthenocarpy 27

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Under our growing conditions all fruits from self-pollinated flowers of the first 29

three inflorescences of WT plants contained seeds. By contrast, in pro plants more than 30

95% of fruits that developed from self-pollinated flowers were parthenocarpic (Table 2 31

and Figs 2A and 2B). Similar results were obtained with D-pro plants (Figs 2A and 2B). 32

Unpollinated pro ovaries had higher weight (Figs 2C and 2D), with larger cells and 33

more cell layers in the pericarp than WT (Fig. 1G). A sudden burst of growth in the 34



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mutant unpollinated ovaries occurred at d3, while unpollinated WT ovaries remained 1

essentially unchanged (Figs 2C and 2D). 2

pro ovaries from emasculated flowers developed parthenocarpically and attained 3

the same final weight as parthenocarpic pro fruits developed under spontaneous self-4

pollination conditions (Table 2 and Fig 2A). This shows that flower emasculation did 5

not affect pro parthenocarpic capacity or the fruit developmental program. Manually 6

pollinated ovaries developed into fruits of higher weight, associated with a higher 7

number of seeds (WT) or to the presence of seeds (pro) (Table 2). This suggests that in 8

the case of pro the developing seeds provide additional factor(s) for fruit development. 9

To know whether parthenocarpy induced by pro was facultative or obligatory we 10

carried out experiments of manual self- and cross-pollination between WT and pro 11

ovaries (Table 3). Fruit-set in all crosses was 100%. Since 60% of the fruits that 12

developed after pro manual self-pollination had seeds, this means that pro 13

parthenocarpy is facultative, and that the absence of seeds under normal culture 14

conditions is probably the result of self-pollination impediment due to the long-style 15

phenotype. The observation that wt x wt and pro x wt fruits (pistil x pollen crosses) had 16

higher number of seeds than wt x pro and pro x pro fruits (Table 3) suggested that the 17

pro mutation reduces male fertility (see also Fig. 1H). This was confirmed by counting 18

the number of extracted pollen grains per anther [17300 ± 4700 in pro vs 55700 ± 3400 19

in WT (n = 10); P < 0.05] (Fig. 1H). However, pollen viability in pro was not reduced, 20

according to red carmine diacetate staining and emission of fluorescence after 21

incubation with fluorescein diacetate. In this case, the lower fluorescence found in pro 22

extracts [385 ±4 vs 586 ± 4 in WT (n = 8); P < 0.01] can be attributed to the reduced 23

number of total pollen grains per extract. Pollen germination capability in the mutant 24

was not reduced either (data not presented). Additionally, the fact that pro x wt fruits 25

had fewer seeds than wt x wt fruits, and pro x pro less than wt x pro indicates that the 26

pro mutation may also affect ovule viability or pollen tube growth in the pro style and 27

ovary. 28

A time-course analysis showed that both cell size (Fig. 3A) and number of cell 29

layers (Fig. 3A and 3B) in the pericarp of pro ovaries was higher than in unpollinated 30

WT ovaries from d-3 to d10, associated with the increase of pericarp thickness (Fig. 31

3C). This indicated that the mutation induces enhancement of cell division and 32

expansion before anthesis and in the absence of pollination. The cell size and number of 33



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cell layers increased from d2 following pollination or GA3 application to unpollinated 1

ovaries at d0, attaining similar values to those of pro from d5 (Fig. 3). 2

Endoreduplication was also monitored to get further insight into the effect of pro 3

on pericarp cytological evolution. Time-course of pericarp cells ploidy was determined 4

both as % of nuclei with different C values (Fig. 3D) and as mean C values (Fig. 3E). 5

The level of endoreduplication was very low and essentially unaltered in WT 6

unpollinated ovaries (with mean C values lower than 3), while steady increase of 7

endoreduplication occurred after d1 in WT pollinated and in unpollinated pro ovaries. 8

At d10, mean C values were about 5 in the last two cases, with values up to 32C in a 9

small percentage of nuclei (Figs 3D and 3E). 10

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Response of procera Plants to GA3 and Paclobutrazol Application 12

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WT tomato plants treated with GA3 display a slender phenotype and have leaves 14

with less serrated borders (Marti et al, 2006), similar to that of pro plants (Figs 4A and 15

4B). This phenotype was obtained after continuous spray application (every two days) 16

of high GA3 dose (10-4 M). The number of leaves before the first inflorescence was also 17

higher in WT plants treated with GA3 (Table 4). Continuous GA3 application to WT 18

plants induced the development of flowers with longer style and protruding stigma (Fig. 19

4C), as well as flowers with increased number of sepals, petals, stamens and carpels 20

(sometimes more than four), similar to pro. In these plants the average fruit weight was 21

similar to pro parthenocarpic fruits and most of them had no seeds (Table 4), probably 22

due to prevention of self pollination by the long style phenotype. Application of GA3 or 23

auxin (IAA or 2,4-D) to pistils of WT emasculated flowers also enhanced style 24

elongation, more the latter than the former (Fig. 4D and corresponding legend). The 25

Brix index value of the juice was higher in parthenocarpic GA3-induced fruits and 26

similar to that of pro fruits, although GA3 application had no effect on fruit-set and 27

growth neither on Brix index in pro plants (Table 4). WT plants continuously treated 28

with GA3 showed many fruits with malformations due to development of one or more 29

additional fruit-like structures at the style end of the fruit, similar to those found in pro 30

(Fig. 4E). When GA3 application was carried out only from flowering onset that kind of 31

fruit malformation did not occur in WT but was enhanced in pro plants (data not 32

presented). 33



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It is known that pro plants have a mutation in the gene encoding the SlDELLA 1

protein, a repressor of GA mode of action (Bassel et al, 2008). This suggests that the 2

observed pro phenotypes are the result of a GA constitutive response induced by this 3

mutation. As expected for a constitutive GA response mutant, pro plants were more 4

resistant than WTs to the reduction of the shoot length and flower organs size by PAC 5

(an inhibitor of GA biosynthesis) (Table 4, Figs 4C and 4F, and Fig. S1). 6

Parthenocarpic fruit development can be induced in tomato by application of GA 7

or auxin (see Serrani et al, 2007a and references therein). Fruits developed from 8

unpollinated pro ovaries were of equal size to unpollinated WT ovaries induced by GA3 9

application, and GA3 did not have additional effect on pro ovaries (Table S1), indicating 10

that fruit-set and growth response to GA3 was saturated in the mutant. By contrast, 11

auxin application to unpollinated pro ovaries enhanced fruit growth, although final 12

weight was lower than that of auxin-treated WT ovaries (Table S1). Therefore, the 13

additional growth response observed in seedy compared to seedless pro fruits presented 14

in Table 3 may be due to auxin provided by the developing seeds. 15

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The procera Mutation Causes Substantial Transcriptome Remodelling in the 17

Ovary 18

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Since we wanted to detect early changes in transcript levels that may be involved 20

in parthenocarpy induction, a comparative transcriptome analysis was performed with 21

unpollinated WT and pro ovaries 3 days before anthesis, a stage at which they had 22

slightly but significantly different weight (Figs 2C and 2D). Those ovaries were mainly 23

constituted of sporophytic tissues, as they contained non-fertilized ovules (Fig. 1G). 24

Transcriptome analysis showed significant and remarkable differences in gene 25

expression in pro compared to WT ovaries. In pro ovaries, 2776 unigenes were 26

differentially expressed, 1346 up-regulated (Table S2) and 1430 down-regulated (Table 27

S3). Similarity to Arabidopsis proteins could be assigned to 88.2% of the differentially 28

expressed sequences. For the 617 genes showing at least two-fold change expression in 29

pro ovaries that were annotated (325 up-regulated, Table S4; 292 down-regulated, Table 30

S5), their functional role was examined using the Munich Information Center for 31

Protein Sequences (MIPS; http://mips.gsf.de) and FunCAt, to search for the most 32

similar corresponding Arabidopsis protein. In most categories, similar proportions of 33

genes were found in both cases (Table S6). 34



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To unveil possible key processes that were altered in pro ovaries, we looked for 1

functional enrichment in the differentially expressed set of genes using the AmiGO tool 2

(http://amigo.geneontology.org) based on the Gene Ontology (GO) terms for the most 3

similar Arabidopsis proteins. Biological Processes identified as most significantly 4

overrepresented (P < 10-5) among the up-regulated genes (Fig. S2A and Table S7) were 5

“Photosynthesis” (GO:0019684) and “Response to stimulus” (GO:0050896), and 6

“Response to hormone stimulus” (GO:0009725) and “Response to endogenous 7

stimulus“ (GO: 0009719) among the down-regulated genes (Fig. S2B and Table S8). 8

Within Cellular Components, the GO category corresponding to “Chloroplast thylakoid 9

membrane” (GO:0009535) was also overrepresented in the up-regulated set. 10

In addition to the genes included in the enriched categories described above, 11

some involved in Cell Organization and in Cell Wall Degradation were up-regulated or 12

down-regulated at least two-fold in pro ovaries (Table S9). Also, the expression of two 13

genes encoding putative peroxidases was altered, one of them up-regulated 14

(homologous to At1g14550) and other (homologous to At5g05340) down-regulated. 15

This is of interest because peroxidase activity has been inversely correlated with cell 16

expansion in pro stem tissues (Jupe and Scott, 1992). In the TF category, 19 genes were 17

significantly up-regulated and 27 down-regulated in pro ovaries, with at least two-fold 18

differential expression (Table S9). In both groups we identified TF from Myb, bHLH, 19

HD-Zip, WRKY, ERF, NAC, and GRAS families. However, MADS-box and bZIP 20

encoding genes (2 and 1, respectively) were only found within those up-regulated, and 21

C2/H2 and Aux/IAA and ARF (2, 1 and 3, respectively) only within those down-22

regulated. 23

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Quantification of Genes Involved in Cell Division and Expansion 25

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We analyzed by qPCR transcript levels of several genes involved in cell division 27

(SlCDKB2.1, encoding a cyclin-dependent kinase; SlCycB2.1, encoding a mitotic 28

cyclin, and SlCycD3.1, encoding a G1 cyclin) and cell expansion (SlEXP5 and 29

SlEXP18, encoding expansins, and SlXTH1 and SlXTH9, encoding xyloglucan 30

endotransglucosylase/hydrolases, XTH), two processes found to be enhanced in pro 31

ovaries even before anthesis (Fig. 3). Those genes belong to large member families, 32

were not present in the array, and were selected because it has previously been reported 33

to show early maximum increase after pollination induced fruit-set (Joubès et al, 2000, 34



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2001; Vriezen et al, 2008). The expression of the three cell division genes was was 1

relatively low in unpollinated WT and pro ovaries before and at the time equivalent to 2

anthesis (d0), and started increasing in pro ovaries from d3 (time of active cell 3

division), with a maximum at d5 and decreasing afterwards (Figs 5A, 5B and 5C). 4

Similar results to pro were found in WT GA3-treated ovaries (Figs 5A, 5B and 5C). 5

Expression of the selected SlEXP and SlXTH cell expansion genes was always low in 6

WT unpollinated ovaries, whereas in the case of pro and WT GA3-treated ovaries it 7

increased steadly after d3 up to at least d10 (Figs 5D, 5E, 5F and 5G), associated with 8

the begining of rapid cell expansion (Fig. 3A). Similar results were found in pollinated 9

and WT unpollinated GA3-treated ovaries, except for SlXTH9 that decreased at d10 10

((Figs 5D, 5E, 5F and 5G). 11

A 450 bp promoter deletion that results in the down-regulation of SlStyle2.1 12

gene (encoding an atypical bHLH protein lacking of DNA binding activity) in 13

developing styles has been reported to reduce style length and favor self-pollination 14

(autogamy) in cultivated tomatoes (Chen et al, 2007). We have confirmed that Micro-15

Tom also contains this SlStyle2.1 promoter deletion (Fig. S3A). SlStyle2.1 transcript 16

levels in pro styles were lower than in WT, although slightly increased in the ovary 17

(Fig. S3B). Moreover, application of 2,4-D but not GA3 to pistils of WT emasculated 18

flowers induced a discrete increment on the accumulation of SlStyle2.1 transcripts (Fig. 19

S3C). Overall, these results suggest that SlStyle2.1 is not responsible for the long style 20

with elongated cells phenotype of pro flowers, 21

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Quantification of Genes Involved in Hormone Signalling and Metabolism 23

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The results of transcriptome analysis showed that the expression of some genes 25

of GA and ABA metabolism, as well as several genes involved in the auxin signalling 26

pathway, was altered in d-3 (three days before anthesis) pro ovaries. This suggests that 27

the pro mutation affects the expression of genes of several hormone pathways, and 28

prompted us to further investigate the extent of this effect by firstly analyzing the 29

expression of the main tomato genes of GA metabolism (Serrani et al, 2007b) and 30

SlDELLA, and ABA metabolism in d-3, d0 and d3 pro and WT ovaries by qPCR (Fig. 31

6). 32

Transcript content of SlGA20ox1, encoding the main GA20ox in tomato ovary, 33

and SlGA20ox2 was enhanced (Fig. 6A), while that of SlGA2ox1, -2 and -4 was reduced 34



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(Fig. 6B) in pro. This was surprising because the expression of those genes has been 1

reported to be respectively under negative and positive GA-feedback regulation, by 2

DELLAs (Yamaguchi, 2008). In the case of GA3ox (also expected to be under GA 3

negative feedback regulation), the expression of SlGA3ox1 was similar in WT and pro, 4

while that of SlGA3ox2 was down-regulated in pro ovaries, suggesting that this gene is 5

under constitutive negative feedback regulation in the mutant (Fig. 6C). Transcript 6

levels of SlDELLA were higher in pro ovaries at the three stages investigated (Fig. 6D), 7

indicating that expression of this repressor is upregulated by the mutation in very young 8

ovaries. Treatment of 7-d-old seedlings with GA3 and PAC confirmed the existence of 9

negative feedback regulation of SlDELLA expression in tomato (Fig. S4). Two genes of 10

ABA metabolism encoding 9-cis-epoxy-carotenoid dioxygenase and a putative (+)ABA 11

8´-hydroxylase, previously shown to regulate ABA content in tomato ovaries (Nitsch et 12

al, 2009), were investigated. However, only in the first case some reduction of 13

expression in pro ovaries was observed (Fig. S5). 14

To estimate the effect of the pro mutation on auxin-signalling, in relation to 15

parthenocarpic growth, we concentrated on two genes previously described to act as 16

repressors of ovary growth not present in the microarray: SlIAA9 (belonging to the 17

Aux/IAA family) (Wang et al, 2005), and SlARF7 (De Jong et al, 2009b). The time-18

course analysis showed that transcript levels of SlIAA9 in unpollinated pro ovaries were 19

very low between d-2 and d3, but increased afterwards, with a maximum at d5 (Fig. 7). 20

The content of SlIAA9 transcripts in WT unpollinated ovaries was always very low, 21

except at d0, which was higher than in pro. WT unpollinated ovaries treated with GA3 22

displayed a similar pattern than pro ovaries (Fig. 7). By contrast, expression of SlARF7 23

was always lower in pro than in WT unpollinated ovaries (between d-2 and d10), and 24

GA3-treatment also dramatically reduced SlARF7 transcript content in WT ovaries (Fig. 25

7). The expression of other genes in the auxin-signalling pathway (SlIAA1, SlIAA3, 26

SlIAA14 and SlARF1), previously shown to display some variation in response to ovary 27

GA3 application (Serrani et al., 2008), was also investigated, but no clear effect of pro 28

on their expression was found (results not presented). 29

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DISCUSSION 31

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Longer style of pro flowers is not controlled by SlStyle2.1 33



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WT tomato species, like S. pennellii, have long styles with stigmas exserted 1

beyond the staminal cone, thus preventing self-pollination. Shorter style length is 2

associated, together with loss of self-incompatibility, with the evolution of autogamy in 3

cultivated tomatoes (Chen et al, 2007). Down-regulation of SlStyle2.1 expression due to 4

a 450 bp promoter deletion (-4656 to -4002) has been associated with the transition 5

from cross-pollination to self-pollination. Micro-Tom also contains that promoter 6

deletion, although some transcripts are still present, as was previously found by in situ 7

hybridization in styles of flowers from S. lycopersicum cv M82 (Chen et al, 2007). 8

However, our results do not support the idea that SlStyle2.1 is responsible for the long 9

style phenotype of pro flowers because its transcript levels were not enhanced in the 10

mutant or in the WT after GA3 application. Nevertheless, since auxin application 11

induces style elongation and accumulation of SlStyle2.1 transcripts in WT pistils, we 12

cannot discard that auxins may play a role in style elongation of pro flowers. 13

The protein encoded by SlStyle2.1 belongs to the atypical bHLH family lacking 14

DNA binding activity (Chen et al, 2007). Several proteins from this family have been 15

shown to regulate hormone-induced cell elongation in Arabidopsis and rice. For 16

instance, PRE1 over-expression in the gai-1 genetic background (bearing a GA 17

insensitive allele of the GAI DELLA protein) suppresses its short hypocotyl phenotype, 18

suggesting that PRE1 may acts as positive regulator of GA signaling presumably 19

downstream of DELLA proteins (Lee et al, 2006). Transcript accumulation of 20

SPATULA (a DNA-binding bHLH gene), involved in style and stigma growth during 21

gynoecium development in Arabidopsis, is negatively regulated by DELLA 22

(Groszamann et al, 2011). Also, DELLA proteins are known to interact with several 23

phytochrome-interacting factors (PIFs; members of a subfamily of DNA-binding bHLH 24

proteins) involved in GA regulation of plant growth and development (Leivar and 25

Quail, 2011). Increase of style length upon SlDELLA depletion in antisense-SlDELLA 26

plants has previously been reported (Marti et al, 2007). Overall, these results suggest a 27

scenery in which reduction of style cell elongation of tomato flowers may occur by 28

SlDELLA sequestering in an inactive complex a transcriptional regulator related to 29

STYLE2.1, through protein-protein interaction. 30

31

procera Flowers Display Meristic Alterations 32

33



15

The high number of pro flowers carrying additional floral organs, and the 1

presence of fruits with additional fruit-like structures was surprising. We also found a 2

similar effect in WT plants continuously treated with GA3, but not when GA3 was 3

applied only just at the time of flowering, probably because this effect of GA3 occurs in 4

the flower meristem at the time of early flower organ formation. Previous reports 5

suggested that the increased number of floral organs in tomato plants grown under low 6

temperature conditions is mimicked by GA3 application (Sawhney, 1983), associated 7

with alteration in MADS-box gene expression (Lozano et al, 1998). The size of the 8

additional fruit-like structures developed in pro fruits was smaller than in WT fruits 9

from plants continuously treated with GA3, but was enhanced in pro plants by GA3 10

application. This may be because the mutated SlDELLA still displays some repressor 11

activity, thus preventing the development of additional carpels, while continuous GA3 12

application may be more efficient to induce endogenous SlDELLA degradation in WT 13

plants. 14

Patterning of the four flower whorls is controlled by the coordinated expression 15

of at least three sets of floral organ identity genes at initiation of floral organ primordia 16

(Ng and Yanofsky, 2001). However, although the eventual fate of organ primordia is 17

determined by the organ identity genes, the arrangement of the floral meristem in 18

concentric whorls, each one with a particular number of units is thought to be regulated 19

by auxin (Cheng and Zhao, 2007). The altered expression of genes encoding several 20

Aux/IAA and ARFs in pro ovaries indicates that some of them may be involved in the 21

observed flower meristic transformations. 22

23

procera Induces Strong Parthenocarpic Capacity Associated with Ovary 24

Transcriptome Remodelation 25

26

The pro ovaries have the capacity to develop parthenocarpically. The absence of 27

seeds was due to the long-style phenotype, which prevents self pollination. The reduced 28

number of pollen grains found in pro flowers is of interest because loss of DELLA 29

repression in rice and barley also leads to less pollen production (see review of Plackett 30

et al, 2011). This means that succesful reproductive development needs restriction of 31

GA response. A long-style phenotype, but not reduced pollen number, was found in 32

tomato antisense-SlDELLA plants (Martí et al, 2007). However, pro parthenocarpy is 33

facultative because fruits with a certain number of seeds could be obtained by manual 34



16

self-pollination. Pro ovaries from emasculated flowers also developed 1

parthenocarpically, while in antisense-SlDELLA plants poor parthenocarpic fruit growth 2

occurs in emasculated flowers. This means that the pro mutation activates a pollination-3

independent developing program in the ovary even under conditions (flower 4

emasculation) where this program is still repressed in antisense-SlDELLA ovaries, 5

probably because pro mutation has a stronger effect on reduction of SlDELLA level or 6

activity than antisense-SlDELLA knock-down mutation. That activation occurs very 7

early in pro, at least 3 days before anthesis, as shown by enhanced weight, larger and 8

more cells, and transcriptome changes in the ovary. 9

The strong parthenocarpic capacity of pro ovaries described before was found 10

under different growing conditions (in the greenhouse throughout the year, using the 11

conditions described in Material and Methods, and in a growth chamber using artificial 12

lighting only), and is not due to the absence of brassinosteroids, because similar 13

phenotypes (including exserted stigmas) were found in D-pro plants. The high 14

penetrance and expressivity of this phenotype was probably not caused by the presence 15

of the sp (short-pruning) mutation in Micro-Tom either, because it has been described 16

that many parthenocarpic fruits also develop from non-emasculated flowers in 17

antisense-SlDELLA of UC82 (a determinate cultivar). The possibility that other 18

mutations present in Micro-Tom (e.g. miniature, mnt) might affect the pro phenotype is 19

unknown. 20

Tomato fruit exocarp constitutes a constraint to turgor-driven growth (Andrews 21

et al, 2002). Cell wall located peroxidases are involved in growth restriction (Passardi et 22

al, 2004) and seem to play an important role in tomato fruit growth regulation (Andrews 23

et al, 2002). Thus, the strong down-regulation of a peroxidase in pro ovaries is of 24

interest in relation to its early parthenocarpic growth. Reduction of peroxidase activity 25

was found in elongating stem tissues of pro and GA3-treated WT tomato plants (Jupe 26

and Scott, 1992), as well as in the la cry pea mutant (Jupe and Scott, 1989; Weston et al, 27

2008). On the other hand, pro mutation up-regulated CUT1, a gene involved in cuticle 28

biosynthesis, which may help to prevent water loss and facilitate ovary growth. Up-29

regulation of genes encoding wax and cuticle biosynthesis enzymes occurs in transgenic 30

citrus with elevated GA (Huerta et al, 2008). 31

Enhanced expression of genes involved in photosynthesis and carbon utilization 32

has been reported after GA application and in citrus overexpressing CcGA20ox1, 33

associated with the increase of net photosynthesis in the leaves (see Huerta et al, 2008). 34



17

In the case of pro ovaries, although net photosynthesis has not been determined, 1

transcript increase of genes encoding proteins involved in light and dark reactions may 2

be relevant to its parthenocarpic growth capacity. 3

4

SlDELLA Modulates Cell Cycle and Expansion Genes During Fruit-Set and 5

Development 6

7

Fruit development in tomato, after pollination, is characterized by a cell division 8

phase (up to about 10 days post anthesis) followed by cell expansion (Gillaspy et al, 9

1993). Unpollinated pro ovaries had more and larger cells than WT, indicating that both 10

processes are stimulated by pro mutation in the absence of pollination and fertilization. 11

Higher number of cell layers was found in pro ovaries as early as three days before 12

anthesis. The complex cell division cycle is controlled by the ordered action of cyclin-13

dependent kinases (CDK) and their positive regulatory proteins named cyclins (Cyc). 14

Thus, the higher transcript level content of SlCDKB2.1 in pro ovaries from at least two 15

days before anthesis, and that of SlCycB2.1 and SlCycD3.1 from d3, supports the 16

hypothesis that the pro positive effect on cell division is mediated, at least partially, by 17

that kinase and cyclins. Increase expression of those genes during cell division phase 18

also occurs in pollinated ovaries (Joubès et al, 2000, 2001; Czerednik et al, 2011) and 19

in parthenocarpic fruits induced in RNAi SlARF7 plants (De Jong et al, 2011). By 20

contrast, in antisense-SlDELLA plants no increase of cell division was found in the fruit 21

(Martí et al, 2007), suggesting that in this case parthenocarpic growth occurs bypassing 22

the cell division phase, This may mean that the residual SlDELLA still present in the 23

antisense plants is still capable of repressing cell divisions in the ovary, an effect that 24

can explain the lower parthenocarpic capacity of antisense-SlDELLA compared to pro 25

plants. On the other hand, EXP and XTH mediate changes in cell wall loosening, and 26

expression of genes encoding both kind of proteins increases in tomato ovaries after 27

pollination (Vriezen et al, 2008) and in RNAi SlARF7 parthenocarpic fruits (De Jong et 28

al, 2011). The higher transcript levels of SlEXP5, SlEXP18, SlXTH1 and SlXTH9 in pro 29

ovaries provide a molecular basis for the positive effect of this mutation in cell 30

expansion. 31

High levels of endopolyploidy occurs in tomate during fruit development 32

(Chevalier et al, 2011). We found that increase of mean C levels started at d1-d2 in pro 33

and WT pollinated ovaries, associated with the first signs of cell expansion, compared 34



18

to the absence of endoreduplication and cell expansion in WT unpollinated ovaries. No 1

difference in the time and degree of endoreduplication between WT pollinated and pro 2

ovaries was observed, at least up to d10. Our results suggest that SlDELLA may act as a 3

repressor of endoreduplication, thus preventing cell expansion. However, since it has 4

been shown that cell and fruit size can be uncoupled from DNA ploidy levels, 5

endoreduplication (both in WT and pro) may act just as a limiting factor for cell 6

expansion during fruit growth (Chevalier et al, 2011). 7

8

Hormone Response and Metabolism Are Altered in the procera Ovary 9

10

The higher SlDELLA transcript content in pro ovaries and seedlings indicates 11

that the gene was subject to negative feedback regulation by its functional protein 12

product in tomato. This agrees with SlDELLA up-regulation in pro leaves (Bassel et al, 13

2008) and in WT ovaries after GA3 application, expected to reduce SlDELLA content 14

(Serrani et al, 2008;). By contrast, only slight or no feedback regulation of DELLA 15

genes occurs in Arabidopsis, rice and barley (Sun and Gubler, 2004). 16

Enhanced expression of SlGA20ox1, which seems to play a pivotal role in 17

tomato fruit-set (Marti et al, 2007; Olimpieri et al, 2007; Serrani et al, 2007b), also 18

occurs in pro ovaries. This was surprising, mainly because feedback regulation of that 19

gene occurs in antisense-SlDELLA (Marti et al, 2007), and in GA3-treated WT tomato 20

ovaries (Martí et al, 2010). Other dioxygenases (e.g. SlGA20ox2 and SlGA2ox2) were 21

not subject to feedback regulation in pro ovaries either. In Arabidopsis, high GA 22

content is associated with enhanced expression of AtGA3ox2 during germination 23

(Frigerio et al, 2006), and of AtGA20ox and AtGA3ox in etiolated compared to 24

deetiolated seedlings (Alabadí et al, 2008). This suggests that feedback regulation of 25

some GA dioxygenases may be uncoupled of GA mode of action in some circumstances 26

to secure a high level of active GAs. 27

Induction of parthenocarpic growth in pea, Arabidopsis and tomato by auxin is 28

mediated in part by GAs, through regulation of GA metabolism (Ozga and Reinecke 29

2003; Serrani et al, 2008; Dorcey et al, 2009). We provide here new information on the 30

auxin-GA cross-talk during fruit-set by showing that GA activity modulates the 31

expression of a gene in the auxin signaling pathway (SlARF7), previously reported to 32

act as a repressor of ovary growth. The pro mutation produced downregulation of 33

SlARF7 expression in the ovaries from before anthesis up to d10, and a similar effect 34



19

was observed upon GA3 application to WT unpollinated ovaries. Lower transcript 1

content of SlARF7 in the cell wall of GA3-treated unpollinated ovaries during the three 2

days after treatment, as well as in pollinated ovaries, has been found before (De Jong et 3

al, 2009b). All these results support the conclusion that SlDELLA upregulates 4

transcription of SlARF7, which encodes a repressor of cell expansion (De Jong et al, 5

2011). By contrast, transcript content of SlIAA9 was low in unpollinated ovaries, 6

showing a small transient maximum peak in WT ovaries at d0. Scarce information on 7

SlIAA9 expression in relation to fruit-set has been reported before. It is of interest that in 8

situ hybridization analysis has shown that pollination triggers the initiation of fruit 9

development associated with the release of a tissue-specific gradient of SlIAA9 10

expression established during flower development (although no quantitative data on 11

SlIAA9 transcript content were reported) (Wang et al, 2009). A proposed model 12

showing the control of fruit-set and development by auxin and GAs, mediated by 13

SlDELLA and the auxin signaling factor SlARF7, based on results presented in this 14

work, and previously by other authors is given in Fig. 8. 15

In summary, our results with pro unveiled new roles of SlDELLA in the control 16

of flower morphology, and ovary cell division and expansion. They also suggest that a 17

transcriptional regulator similar to SlStyle2.1, encoding an atypical bHLH, is a possible 18

target of that GA-repressor. The results showed a new kind of GA-auxin cross-talk 19

during fruit-set, where auxin and GA activities regulate the expression of SlARF7 20

encoding a repressor of ovary growth, and support the idea that fruit-set and 21

development is controlled by a complex regulatory gene network. 22

23

MATERIALS AND METHODS 24

25

Plant Material 26

27

Near isogenic lines of tomato (Solanum lycopersicum L. cv Micro-Tom) WT, 28

procera (pro), Dwarf (D) and introgressed D-pro, and S. pennellii (accession LA0716, 29

TGRC, University of Davis, USA) were used in the experiments. The pro allele was 30

introgressed in Micro-Tom (BC6F2) from LA0565 cv Condine Red, and D (a WT gene 31

encoding 6-deoxocatasterone oxidase) from cv Manyel, as described in Carvalho et al 32

(2011). The plants were grown in a greenhouse under 24ºC (day)/20ºC (night) 33

conditions, in 0.6 L pots with peat:vermiculite (1:1), and irrigated with Hoaglands´s 34



20

solution. Natural light was supplemented with Osram lamps (Powerstar HQI-BT, 1

400W) to get a 16 h light photoperiod. Two-three flowers per truss and the first two-2

three trusses were normally used for the experiments. Flower emasculation was carried 3

out two days before anthesis (d-2) to prevent self-pollination. Cross-pollination was 4

carried out at d0 on previously emasculated flowers. All non-selected flowers were 5

removed. 6

7

Application of Plant Growth Substances 8

9

GA3 (10-5 and 10-4 M) was sprayed to the entire plants as aqueous 0.1% Tween 10

20 solution. PAC (10-5 and 10-4 M) was sprayed or applied to the roots in the nutrient 11

solution. GA3 (2000 ng), IAA (2000 ng) and 2,4-D (200 ng) were also applied to 12

unpollinated ovaries from previously emasculated flowers in 10 µL of 5% ethanol, 0.1% 13

Tween 20 solution. All plant growth substances were from Duchefa (Haarlem, The 14

Netherlands). Control plants and ovaries were treated with the same volumes of solvent 15

solutions. 16

17

Histology 18

19

Tissue sections of d-2, d0, d3, d5 and d10 ovaries were fixed in 4% 20

paraformaldehyde 0.1 M sodium phosphate buffer pH 7.2. After dehydration in ethanol 21

the samples were embedded in paraffin (Paraplast Plus, Sigma-Aldrich). Microtome 8 22

µm thick sections were stained with Safranine-Alcian blue solution (mixture of 2 mL 23

0.1% Safranine in 50% ethanol and 5 mL 0.1% Alcian blue in 50% ethanol, diluted in 24

200 mL of 0.1 M acetate buffer pH 5.0), viewed on a microscope and photographed 25

with a spot digital camera (DMX1200F, Nikon). 26

Cell size was estimated by measuring the longest diameter of 10 cells in 27

transversal sections from at least four fruits, anthers and styles (one section per organ). 28

In the case of pericarp, only cells from internal mesocarp were considered. Number of 29

cell layers was determined by counting the number of cells along a line across the 30

pericarp perpendicular to epidermis and endocarp (avoiding vascular vessels). 31

32

Ploidy Determination 33

34



21

Ploidy of d-2 to d10 WT ovaries was determined as described before (Serrani et 1

al, 2007a). In the case of WT pollinated and pro d3 to d10 developing fruits, only the 2

pericarp was used. Briefly, the material was sliced with a razor blade into 0.4 mL of 3

nuclei isolation buffer (High resolution DNA kit solution A: nuclei isolation, Partec 4

GmbH, Münster, Germany), mixed with 1 mL of staining buffer (“High resolution DNA 5

Kit”, solution B: DAPI staining; Partec GmbH, Münster, Germany), shaken for 1 min 6

and filtered through 100 µm nylon mesh (Nyblot). The filtrates (more than 5000 nuclei 7

per extract) were analyzed using a Partec PA-II flow cytometer (Partec GmbH). Peak 8

areas corresponding to nuclei with different ploidy, taking young C values as reference, 9

were used to calculate nuclei percentages distribution. Mean C values (MCV) 10

correspond to the sum of the number of nuclei of each ploidy level multiplied by its 11

endoreduplication cycle, divided by the total number of nuclei (Barow and Meister, 12

2003). 13

14

Pollen Quantification and Viability 15

16

Anthers were extracted with a Vortex in Eppendorf vials with 500 μl of 0.5 M 17

sucrose solution. The pollen was pelleted and resuspended in 200 μl, deposited on a 18

microscope slide, and the number of pollen grain counted in 20 randomly selected field 19

trials per slide. 20

Pollen viability was determined using two staining methods. First, looking at the 21

number of pollen grains stained in red with carmine acetate solution. Second, by 22

incubating extracted pollen in 2.4 x 10-5 M fluorescein diacetate solution for 10 min, and 23

measuring fluorescence intensity (λex = 494 nm, λem = 521 nm) in a Perkin-Elmer 24

LS50B spectrofluorimeter (Pinillos et al, 2008). 25

Pollen germination tests were performed on glass slides coated with germination 26

medium [0.292 M sucrose, 1.27 mM Ca(NO3)2, 1.62 mM H3BO3, 1mM KH2PO4 and 27

0.5% agarose]. The number of germinated pollen grains was counted under microscope 28

after 2 h incubation at 25ºC in the dark. 29

30

Transcriptome Analysis 31

32

Transcript profiles corresponding to WT and pro d-3 ovaries were compared 33

using the tomato 70-mer oligo microarray (EU-TOM2) containing 11857 unigenes, and 34



22

the updated version TomatoCombine#5 (SGN-U5xxxxx) of the tomato unigene 1

database (Bombarely et al, 2011) (http://solgenomics.net/). According to the pre-2

published ITAG2.3 release of the tomato genome annotation, the EU-TOM2 oligo 3

microarray represents 7575 different gene models. 4

Total RNA was extracted using the RNAqueous and Turbo DNA-free kits 5

(Ambion, Applied Biosystems). Labeled cDNA was prepared using 1 µg of total RNA 6

and the Amino Allyl MessageAmpII aRNA amplification kit (Ambion). Cy3/Cy5-7

labeled cDNAs corresponding to three WT and pro independent biological samples with 8

a dye-swap were hybridized to 4 independent array slides. Images were acquired using 9

Genepix 6.0 image acquisition program. Statistical analysis of data was performed with 10

the GeneSpring vs GX 9.0.5 software (Agilent Technologies). Only genes showing at 11

least 2-fold differential expression between samples, with a P value <0.05 in one way 12

ANOVA (parametric test without the assumption of equal variances), were used for 13

further analyses. 14

15

Quantitative PCR 16

17

One µg of total RNA (RNAqueous-4PCR kit and Plant RNA Isolation Aid; 18

Ambion, Applied Biosystems) was used to synthesize first-strand cDNA (TaqMan 19

reverse transcription kit; Ambion). Transcript levels were determined by absolute qPCR 20

according to the methodology described in detail elsewhere (Serrani et al, 2008), using 21

specific primers (see Table S10 and Serrani et al, 2008). Amounts of mRNA in samples 22

were quantified using three biological replicates. 23

24

ACKNOWLEDGMENTS 25

26

We thank Dr A Bombarely (Sol Genomics Networks database) for help with 27

tomato gene annotation, Dr C Gisbert for providing seedlings of S. pennellii, Mrs T 28

Sabater for care of plants in the greenhouse, and Drs D Alabadí and MA Pérez-Amador 29

for critical reading of the manuscript. 30

31

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27

Plackett ARG, Thomas SG, Wilson ZA, Hedden P (2011) Gibberellin control of 1

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6



29

Table 1. Phenotypes of WT and pro mutant. Values are means ± SE. Statistical 1

significance of mean differences was analysed using t-test: *, P < 0.05; **, P < 0.01. 2

3

WT procera

Hypocotyl length (mm)a 14.3 ± 0.3 18.3 ± 0.2** Root length (mm)a 63.8 ± 0.9 58.2 ± 1.4** Height to first inflorescence (cm)b 10.4 ± 0.4 17.2 ± 0.4** Leaves until first inflorescence (n)b 6.6 ± 0.2 8.6 ± 0.2** Stem diameter of fifth internode (mm)b 6.6 ± 0.1 5.0 ± 0.1** Flowers in the two first inflorescences (n)b 12.5 ± 0.2 9.4 ± 1.0* Sepal length (mm)c 6.8 ± 0.3 7.9 ± 0.3** Petal length (mm)c 11.6 ± 0.3 14.0 ± 0.3** Staminal cone length (mm)c 7.4 ± 0.2 8.2 ± 0.2** Style length (mm)c 5.8 ± 0.1 7.7 ± 0.2** Sepals, petals and stamens per flower (n)d 5.0 ± 0.1 5.7 ± 0.2** Carpels per flower d(n) 2.9 ± 0.2 4.0 ± 0.2** Cell size (µm)

- Style - Anther wall - Pericarp

1.82 ± 0.08 4.53 ± 0.30 2.10 ± 0.08

2.43 ± 0.13** 5.31 ± 0.38*

2.98 ± 0.16** Pericarp cell layers (n) 6.9 ± 0.1 10.3 ± 0.2** Days to anthesis of first flower (n)e 38.5 ± 0.1 43.7 ± 0.2** Days to breaker (colour change) of first mature fruite 78.0 ± 0.3 87.5 ± 0.1** Fruits per plant (n)e 24.6 ± 2. 42.5 ± 2.1** Fruit weigh (g)e 5.8 ± 0.3 2.3 ± 0.2** Fruit production (g/plant)e 127.6 ± 16.6 83.8 ± 5.5* Seeds per fruit (n)e 34 ± 3 0 Brix indexf 4.7 ± 0.2 7.2 ± 0.2*

4 a 6 seven-day-old seedlings; b12 full developed plants; c12 random flowers from six 5

plants; d75 random flowers from two independent batches; in the case of pro flowers, 6

42% (27/63) and 66% (8/12) of them, respectively, had six sepals, petals and stamens 7

and four carpels; e12 plants; fBrix index was determined in juice collected from five 8

mature fruits from 12 plants. 9

10

11



30

Table 2. Effect of flower emasculation and pollination on fruit-set and growth. When 1

fruitful, values are means ± SE of 36 fruits from 6 plants, 6 fruits per plant. 2

3

Flower

treatment

WT procera

Fruit-set

(%)

g/fruit Seeds Fruit-set

(%)

g/fruit Seeds

Spontaneous

self-

pollination

100 6.2 ± 1.0 Yes 100 2.3 ± 0.3 No

Emasculated 0 - - 100 2.4 ± 0.3 No

Emasculated +

manual

pollination

100 7.9 ± 0.8 Yes 100 4.4 ± 0.7 Yes

4

5

6



31

Table 3. Effect of WT and pro crosses on fruit-set and growth. Successful fruit-set and 1

number of fruits with seeds versus total number of attempts is recorded. The number of 2

seeds per fruit refers only to fruits with seeds. 3

4 5

Fruit set (fruits developed/ number of attempts)

Seeded fruits (fruits with

seeds/number of attempts)

Weight

(g/fruit)

Seeds

(n/fruit)

♀ wt x ♂ wta 10/10 10/10 5.9 ± 0.4 35.0 ± 3.1

♀ pro x ♂ proa 10/10 0/10 2.5 ± 0.2 0

♀ wt x ♂ wt 10/10 10/10 5.0 ± 0.8 21.1 ± 1.3

♀ pro x ♂ pro 10/10 6/10 4.3 ± 0.5 3.2 ± 0.7

♀ pro x ♂ wt 10/10 10/10 5.5 ± 0.7 16.1 ± 1.8

♀ wt x ♂ pro 10/10 10/10 4.7 ± 0.5 8.7 ± 1.3

6 aSpontaneous self-pollination. 7

8



32

Table 4. Effect of PAC and GA3 application to entire plants on vegetative and fruit-1

growth in WT and procera plants. PAC (10-5 M) and GA3 (10-5 M) were applied after 2

the development of the first true leaf, every two days until flowering. Values are means 3

of 10 plants and 50 fruits. Brix index was determined in juice collected from five 4

mature fruits from 10 plants. PAC, paclobutrazol. 5

6

WT procera

Mock + GA3 + PAC Mock + GA3 + PAC

Height to first inflorescence (cm)

10.4 ± 0.3

11.7 ± 0.3

6.1 ± 0.2

17.2 ± 0.2

17.6 ± 0.2

13.5 ± 0.2

Leaves to 1st inflorescence (n)

6.6 ± 0.2

7.8 ± 0.3

5.6 ± 0.2

8.6 ± 0.2

8.2 ± 0.3

7.8 ± 0.1

Style length (mm) 5.9 ± 0.4

7.0 ± 0.3

4.1 ± 0.3

7.3 ± 0.3

8.8 ± 0.4

5.8 ± 0.3

Days to anthesis of first flower (n)

38.5 ± 0.8

36.6 ± 0.1

40.4 ± 0.5

43.7 ± 0.2

42.2 ± 0.2

42.2 ± 0.2

Fruits per plant (n) 25.4 ±

2.0 29.6 ±

1.2 - 40.5 ±

2.2 39.5 ±

1.5 -

Weight (g/fruit) 5.8 ± 0.5

3.0 ± 0.6

- 3.0 ± 0.4

3.0 ± 0.45

-

Seeds (n/fruit) 21.8 ±

2.6 0.9 ± 1.5

- 0 0

-

Seedless fruits (%) 7.7 ± 3.0

75.3 ± 1.5

- 100 ± 0

100 ± 0

-

Brix index 4.8 ± 0.3

6.8± 0.4

- 7.2 ± 0.2

6.5 ± 0.2

-

7

8



33

FIGURE LEGENDS 1

2

Figure 1. Phenotypes of tomato pro mutant. (A) Seven-day-old seedlings (left) and 3

plants at the time of flowering (right). (B) Length of different internodes. Values are 4

means from six plants ± SE. (C) Full developed WT, pro, D and D-pro plants. (D) 5

Leaves from different positions in the plant (at node 1 to 5 from cotyledons). (E) Entire 6

flowers (two upper rows) and stamens (lower row) from WT, pro, D and D-pro plants. 7

(F) Intact staminal cones showing protruding styles in pro and D-pro flowers. (G) 8

Transversal sections of d-3, d0 and d3 ovaries. (H) Stigma and transversal sections of 9

styles and anthers stained with Safranine-Alcian blue, and extracted pollen stained with 10

Carmine acetate . (I) Close-up of transversal sections of pericarp, anther and style, and 11

longitudinal sections of styles at the time of anthesis. WT, WT (Micro-Tom); pro, 12

procera; D, Dwarf; D-pro, D-introgressed -procera. 13

Figure 2. Growth of WT and pro pistils and fruits. (A) Effect of pollination (self- and 14

hand pollination) and emasculation of WT, pro, D and D-introgressed-pro flowers on 15

final fruit weight. Values are means ± SE of 25 (WT and pro) and 6 (D and D-pro) 16

fruits. (B) Picture of representative fruits developed from flowers subjected to different 17

treatments. Fruits with additional fruit-like structures are also presented in the case of 18

self-pollinated pro and D-pro. (C) Time-course of the weight of unpollinated pistils. 19

Values are means ± SE of three replicates, each one consisting of a mixture of 10 pistils. 20

(D) Picture of representative unpollinated pistils from d-3 to d5. 21

Figure 3. Time-course between d-3 and d10 of cell division and expansion in pericarp 22

of pr , WT pollinated, and in WT unpollinated control and GA3 treated ovaries. (A) 23

Photographs of transversal sections from pro, and WT pollinated ovaries. (B) Number 24

of cell layers. (C) Pericarp thickness. (D) Nuclei frequency. (E) Mean C values. 25

Figure 4. Effect of GA3 and PAC application to WT and pro plants. (A) Plants WT 26

control and treated with GA3 compared to pro plants. (B) Morphology of the fifth leaf 27

(from the cotyledons) of non-treated and GA3-treated plants. (C) Flower morphology 28

and size of pistils from plants treated with GA3 and PAC. (D) Effect of GA3, IAA and 29

2,4-D on style elongation of WT pistils. The pistils were treated with hormone solutions 30

on d-5 after removing stamens and petals, and style length was measured on d0. Final 31

means ± SE of style lengths (mm) were: Mock, 3.8 ± 0.2; +GA3, 4.7 ± 0.1*; +IAA 5.3 ± 32

0.2**; +2,4-D, 6.1 ± 0.2 ** (n = 7)(*, P < 0.05; **, P < 0.01). (E) Effect of continuous 33

GA3 application to plants on morphology of representative fruits displaying additional 34



34

fruit-like structures. (F) Plants WT and pro non-treated and treated with PAC. WT, WT; 1

pro, procera; PAC, paclobutrazol. 2

Figure 5. Time-course between d-2 and d10 of transcript levels of cell division and 3

expansion genes in pro and in WT unpollinated control and GA3 treated ovaries. (A) 4

SlCDKB2.1. (B) SlCycD3.1. (C) SlCycB2.1. (D) SlEXP5. (E) SlEXP18. (F) SlXTH1. (G) 5

SlXTH9. Absolute amount of RNA means molecules of RNA/ng total RNA. 6

Figure 6 Transcript levels of GA metabolism and SlDELLA genes in unpollinated WT 7

and pro ovaries between d-3 and d3. (A) SlGA20ox1, SlGA20ox2 and SlGA20ox4. (B) 8

SlGA2ox1, SlGA2ox2 and SlGA2ox4. (C) SlGA3ox1 and SlGA3ox2. (D) SlDELLA. 9

Absolute amount of RNA means molecules of RNA/ng total RNA. 10

Figure 7. Time-course between d-2 and d10 of SlIAA9 (A) and SlARF7 (B) transcript 11

content in pro and in WT unpollinated control and GA3 treated ovaries. Absolute 12

amount of RNA means molecules of RNA/ng total RNA. 13

Figure 8. Model for auxin and GA interaction through SlDELLA during tomato fruit-14

set and development. Thick lines correspond to previously well established processes, 15

and thin lines to proposed relationships based on results of this work or from other 16

authors. 1, possible auxin-independent regulation of GA metabolism. 2, from De Jong et 17

al (2009b) and unpublished personal data. 3, from De Jong et al (2011). In addition to 18

down-regulation of SlARF7 transcription by auxin, this hormone would be expected to 19

promote the capacity of SlARF7 to regulate gene expression by virtue of auxin-induced 20

degradation of Aux/IAA proteins. 21

22

23



35

Supplemental files 1

Table S1. Effect of GA3 and 2,4-D application to unpollinated WT and pro ovaries on 2

fruit-set and growth (g/fruit). GA3 (2000 ng) and 2,4-D (200 ng) were applied to ovaries 3

from emasculated flowers, and fruits collected at d20. Values are means ± SE of 25 4

treated ovaries. Statistical significance of mean differences was analysed using t-test: *, 5

P < 0.05; **, P < 0.01; NS, not significant. 6

Table S2. Tomato gene annotations of significantly up-regulated genes in pro ovaries. 7

Table S3. Tomato gene annotations of significantly down-regulated genes in pro 8

ovaries. 9

Table S4. Tomato gene annotations of up-regulated genes in pro ovaries with at least 10

two-fold change. Data were extracted from those presented in Table S2, but including in 11

this case tomato annotations. 12

Table S5. Tomato gene annotations of down-regulated genes in pro ovaries with at least 13

two-fold change. Data were extracted from those presented in Table S3, but including in 14

this case tomato annotations. 15

Table S6. MIPS Functional Catalogue analysis of tomato differentially expressed genes 16

in pro ovaries. 17

Table S7. Gene ontology of biological processes categories over-represented in the up-18

regulated set. 19

Table S8. Gene ontology of biological processes categories over-represented in the 20

down-regulated set. 21

Table S9. Differentially expressed genes encoding transcription factors and proteins 22

related to cell division and cell wall biosynthesis and modification. 23

Table S10. Primers used for qPCR. 24

25

Figure S1. (A) Effect of PAC on growth of WT and pro plants (n = 12 plants). (B) 26

Effect of GA3 and PAC on growth of flower organs at anthesis (n = 10 flowers from 27

different plants). The plants were sprayed with PAC (10-4 M) and GA3 (10-4 M) 28

solutions during 4 consecutive weeks after germination. 29

Figure S2. Hierarchical view of GO Biological Processes categories overrepresented 30

with up-regulated (A) and down-regulated (B) genes. Significant categories (P-value 31

from Fisher´s exact test corrected for multiple hypothesis testing <0.05) are shown 32

using a gray colour scale according to their significant level. Other categories required 33

to complete the hierarchy are also shown. 34



36

Figure S3. (A) Amplification of genomic DNA from S. pennellii and S. lycopersicum 1

(cv Micro-Tom) using primers spanning the 450 bp promoter deletion of SpStyle2.1. (B) 2

Transcript levels of SlStyle2.1 in styles and ovaries of WT and pro flowers. (C) Effect 3

of GA3 and 2,4-D application on SlStyle2.1 transcript levels in pistils of WT flowers. 4

The hormones were applied on d-3 to pistils of emasculated flowers and the material 5

was collected four days later. Absolute amount of RNA means molecules of RNA/ng 6

total RNA. 7

Figure S4. Effect of GA3 and PAC on SlDELLA expression in 7-d-old WT seedlings. 8

The seedlings were grown in Petri dishes with solidified medium control (mock) or 9

containing 10-5 M GA3 or 10-5 M PAC. PAC, paclobutrazol. Absolute amount of RNA 10

means molecules of RNA/ng total RNA. 11

Figure S5. Transcript levels of ABA metabolism genes (SlNCD1 and SlCYP107A1) in 12

unpollinated WT and pro ovaries between d-3 and d3. Absolute amount of RNA means 13

molecules of RNA/ng total RNA. 14

15



Fig. 1E

5 cm

A

2 cm

2,5

3,0

3,5

4,0

th (c

m)

1 cm

WT pro D D-pro

WT proWT pro

G d-3 d0 d3

B

1 2 3 4 5 6 7 8 90,5

1,0

1,5

2,0

WT pro

Len

g

Internode

C

WT

pro

H10 cm

C

500 μm

pro

Style

WT

Anther PollenStigma

D D-proWT pro

pro

500 μm100 μm200 μm300 μm

D

WTpro

2 cm

I Pericarp

WT pro

Style

WT pro

F

pro

1 2 3 4 5

WT proAnther

25 μm

WT pro D D-pro

0.5 cm25 μm 25 μm



• Figure 1. Phenotypes of tomato pro mutant. (A) Seven-day-old seedlings (left) and plants at the time of flowering (right). (B) Length of different internodes. Values are means from six plants ± SE. (C) Full developed WT, pro, D and D-pro plants. (D) Leaves from different positions in the plant (at node 1 to 5 from cotyledons). (E) Entire flowers (two upper rows) and stamens (lower row) from WT, pro, D and D-pro plants. (F) Intact staminal cones showing protruding styles in pro and D-proflowers. (G) Transversal sections of d-3, d0 and d3 ovaries. (H) Stigma and transversal sections of styles and anthers stained with Safranine-Alcian blue, and

t t d ll t i d ith C i t t (I) Cl f t l tiextracted pollen stained with Carmine acetate . (I) Close-up of transversal sections of pericarp, anther and style, and longitudinal sections of styles at the time of anthesis. WT, WT (Micro-Tom); pro, procera; D, Dwarf; D-pro, D-introgressed -procera.



Fig. 2

8 A Self-pollinated Emasculated HandpollinatedB

Wei

ght (

g)

0

2

4

6Self-pollinated EmasculatedHandpollinated

WT pro WT pro WT pro

B

8

10

12

14

16

WT pro

Wei

ght (

mg)

C

0 D D-proWT pro

D D-pro D D-pro D D-pro

Wei

ght (

mg)

d-3 d-2 d-1 d0 d1 d2 d3 d52

4

6

Day

WT

D

1cm

d-3 d-2 d-1 d0 d1 d2 d3 d5 Day

pro

Figure 2. Growth of WT and pro pistils and fruits. (A) Effect of pollination (self- and hand pollination) and emasculation of WT, pro, D and D-introgressed-pro flowers on final fruit weight. Values are means ± SE of 25 (WT and pro) and 6 (D and D-pro) fruits. (B) Picture of representative fruits developed from flowers subjected to different treatments. Fruits with additional fruit-like structures are also presented in the case of self-pollinated pro and D-pro. (C) Time-course of the weight of unpollinated pistils. Values are means ± SE of threepro. (C) Time course of the weight of unpollinated pistils. Values are means SE of three replicates, each one consisting of a mixture of 10 pistils. (D) Picture of representative unpollinated pistils from d-3 to d5.



Fig. 3

A

WT proWT pro WT pro WT pro

d5 d10PollUnpoll PollUnpoll

d-2 d-1 d0d-3

d1 d2 d3

WT pro GA3

PollUnpollWTWT pro GA3

PollUnpollWT WT pro GA3

PollUnpollWT

WT pro GA3 WT WT pro GA3 WT

B C

Peri

carp

thic

knes

s (μm

)

200

400

600

800

1000WT unpollinated ovariesWT pollinated ovariesWT GA3-induced ovariesprocera unpollinated ovaries

º of p

eric

arp

cell

laye

rs

10

15

20

25

30WT unpollinated ovariesWT pollinated ovariesWT GA3-induced ovariesprocera unpollinated ovaries

D EDays post-anthesis

-3 -2 -1 0 1 2 3 4 5 6 7 8 9 10

P 200

0

MC

V)

5,5

6,0

6,5

7,0WT unpollinatedWT pollinated procera unpollinated ovaries

y (%

) 80

100

2C4C8C16C32C

Days post-anthesis-3 -2 -1 0 1 2 3 4 5 6 7 8 9 10

N

5

Days post-anthesis-2 -1 0 1 2 3 4 5 6 7 8 9 10

Mea

n C

val

ue (M

2,5

3,0

3,5

4,0

4,5

5,0

,

Nuc

lei f

recu

ency

0

20

40

60

Unpollinated 2 3 4 51

Pollinated procera WT

Unpollinated

2C

10 -2 -1 0 2 3 4 51 10-2 -1 0 2 3 4 51 10

Figure 3. Time-course between d-3 and d10 of cell division and expansion in pericarp of pr , WT pollinated, and in WT unpollinated control and GA3 treated ovaries. (A) Photographs of transversal sections from pro, and WT pollinated ovaries. (B) Number of cell layers. (C) Pericarp thickness. (D) Nuclei frequency. (E) Mean C values.



Fig. 4

A B

WT Mock +GA3 pro

A 2cm

2cm

WT

B

0.5 cmD

C

C +GA3 +PAC

WTMock +GA3

pro

1 cmE

1 cm

pro

WT

F

WT

pro

Mock +GA3

3 cm

Mock +PAC

WT

pro

Figure 4. Effect of GA3 and PAC application to WT and pro plants. (A) Plants WT control and treated with GA3 compared to pro plants. (B) Morphology of the fifth leaf (from the cotyledons) of non-treated and GA3-treated plants. (C) Flower morphology and size of pistils from plants treated with GA3 and PAC. (D) Effect of GA3, IAA and 2,4-D on style elongation of WT pistils. The pistils were treated with hormone solutions on d-5 after removing stamens and petals, and style length was measured on d0. Final means ± SE of st le lengths (mm) ere: Mock 3 8 ± 0 2; +GA 4 7 ± 0 1*; +IAA 5 3 ±means ± SE of style lengths (mm) were: Mock, 3.8 ± 0.2; +GA3, 4.7 ± 0.1*; +IAA 5.3 ±0.2**; +2,4-D, 6.1 ± 0.2 ** (n = 7)(*, P < 0.05; **, P < 0.01). (E) Effect of continuous GA3 application to plants on morphology of representative fruits displaying additional fruit-like structures. (F) Plants WT and pro non-treated and treated with PAC. WT, WT; pro, procera; PAC, paclobutrazol.



Fig. 5

3,5e+5SlCDKB2.1

8e+3SlCycD3.1BA

-2 -1 0 1 2 3 4 5 6 7 8 9 10

Abs

olut

e am

ount

of R

NA

0,0

5,0e+4

1,0e+5

1,5e+5

2,0e+5

2,5e+5

3,0e+5

0

WT unpoll.

WT +GA3procera unpoll.

-2 -1 0 1 2 3 4 5 6 7 8 9 10

Abs

olut

e am

ount

of R

NA

0

2e+3

4e+3

6e+3

0

WT unpoll.

WT +GA3

procera unpoll.

BA

Abs

olut

e am

ount

of R

NA

1e+3

2e+3

3e+3

4e+3

5e+3

6e+3

7e+3SlCycB2.1

WT unpoll

WT +GA3

procera unpoll.

C

Days post-anthesis Days post-anthesis

Abs

olut

e am

ount

of R

NA

2,0e+3

4,0e+3

6,0e+3

8,0e+3

1,0e+4

1,2e+4

1,4e+4

1,6e+4SlEXP5

WT unpoll

WT +GA3

procera unpoll.

D


0 0WT unpoll.


0,0

,0e 3

0

WT unpoll.

mou

nt o

f RN

A

3e+6

4e+6

5e+6

6e+6SlEXP18

WT +GA3

procera unpoll.E

mou

nt o

f RN

A

8 0e+5

1,0e+6

1,2e+6

1,4e+6

1,6e+6

1,8e+6SlXTH1

WT +GA3

F


Abs

olut

e a

0

1e+6

2e+6

0

WT unpoll.


Abs

olut

e am

0,0

2,0e+5

4,0e+5

6,0e+5

8,0e+5

0WT unpoll.

procera unpoll.

A 2 0e+5

2,5e+5SlXTH9G


Abs

olut

e am

ount

of R

NA

0,0

5,0e+4

1,0e+5

1,5e+5

2,0e+5

0

WT unpoll.

WT +GA3

procera unpoll.

Figure 5. Time-course between d-2 and d10 of transcript levels of cell division and expansion genes in pro and in WT unpollinated control and GA3 treated ovaries. (A) SlCDKB2.1. (B) SlCycD3.1. (C) SlCycB2.1. (D) SlEXP5. (E) SlEXP18. (F) SlXTH1. (G) SlXTH9. Absolute amount of RNA means molecules of RNA/ng total RNA.



Fig. 6A

bsol

ute

amou

nt o

f RN

A

2 5e+4

5,0e+4

7,5e+4

1,0e+5

1,3e+5A

Abs

olut

e am

ount

of R

NA

5,0e+4

1,0e+5

2,0e+5 B

Abs

olut

e am

ount

of R

NA

A

0,0

2,5e+4

WT pro

SlGA20ox1

WT pro

SlGA20ox2

WT pro

SlGA20ox4

1,0e+5

4,0e+5 C2,0e+5

d-3 d0 d3

D

A

0,0WT pro

SlGA2ox1

WT pro

SlGA2ox2

WT pro

SlGA2ox4

RN

A

0,0

2,5e+4

5,0e+4

7,5e+4

WT pro WT pro0,0

5,0e+4

1,0e+5

1,5e+5

WT pro

Abs

olut

e am

ount

of R

Figure 6. Transcript levels of genes of GA metabolism andsignalling in unpollinated WT and pro ovaries between d-3 and d3.(A) SlGA20ox1, SlGA20ox2 and SlGA20ox4. (B) SlGA3ox1 andSlGA3 2 (C) SlGA2 1 SlGA2 2 d SlGA2 4 (D) SlDELLA

p

SlGA3ox1

p

SlGA3ox2 SlDELLA

SlGA3ox2. (C) SlGA2ox1, SlGA2ox2 and SlGA2ox4. (D) SlDELLA.Absolute amount of RNA means molecules of RNA/ng total RNA.



Fig. 7A 5e+6

6e+6SlIAA9

A 2,5e+5

3,0e+5SlARF7

WT unpoll.A B

Fig. 7

2 1 0 1 2 3 4 5 6 7 8 9 10

Abs

olut

e am

ount

of R

NA

0

1e+6

2e+6

3e+6

4e+6

5e+6

0WT unpoll.

WT +GA3

procera unpoll.

-2 -1 0 1 2 3 4 5 6 7 8 9 10

Abs

olut

e am

ount

of R

NA

0,0

5,0e+4

1,0e+5

1,5e+5

2,0e+5

2,5e 5

0

WT unpoll.

WT +GA3

procera unpoll.

Figure 7. Time-course between d-2 and d10 of SlIAA9 (A) and SlARF7 (B) transcript content in pro and in WT unpollinated control and GA3 treated ovaries. Absolute amount of RNA means molecules of RNA/ng total RNA.


Days post-anthesis2 1 0 1 2 3 4 5 6 7 8 9 10



Fig. 8

Fig. 8. Model for auxin and GA interaction through SlDELLA duringtomato fruit-set and development. Thick lines correspond to previouslywell established processes, and thin lines to proposed relationshipsbased on results of this work or from other authors. 1, possible auxin-independent regulation of GA metabolism. 2, from De Jong et al(2009b) and unpublished personal data. 3, from De Jong et al (2011).In addition to down-regulation of SlARF7 transcription by auxin, thishormone would be expected to promote the capacity of SlARF7 toregulate gene expression by virtue of auxin-induced degradation ofAux/IAA proteins.

1

2

Pollination/Fertilization

FruitFruit--set andset anddevelopmentdevelopment

GAs

SlDELLA

CellCell expansionexpansion

Auxin

CellCell divisiondivision

SlARF7

3 3



1 1 2 3 4 5 6 Running head: Tomato procera - Plant Physiology

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