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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
  • C12N15/823—Reproductive tissue-specific promoters
  • C12N15/8233—Female-specific, e.g. pistil, ovule
• • C—CHEMISTRY; METALLURGY
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8237—Externally regulated expression systems
  • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• the present invention is related to a method for adjusting the shape of plant cells and plant organs.
• the invention is also related to GEG cDNA or GEG-like nucleic acid sequences and their products useful in horticulture, agriculture and forestry for manufacturing renewable plant-derived raw-materials with structures more convenient, advantageous and feasible for the respective application.
• the objectives of the present invention is to provide new methods for altering or adjusting the shape of plant cells and organs by controlling and/or regulating the direction, both in longitudinal and radial direction, as well as the magnitude of cell growth.
• Another objective of the present invention is to provide means carrying out said method.
• the means are GEG cDNA or GEG-like nucleic acid sequences, including promoters of said GEG or GEG-like genes or nucleic acid sequences capable of directing foreign genes or heterologous nucleic acid sequences in desired direction.
• the GEG-like gene products of said nucleic acid sequences are useful in agriculture, horticulture and forestry for manufacturing renewable plant-derived raw-materials with structures more convenient, advantageous and feasible for the respective application.
• Especially preferred objectives of the present invention in forestry is to enable modification of the length and/or breadth of the fibers obtainable for example from wood.
• one of the desired objectives is to provide shorter and broader stems which are more stable or resistant to crop flattening by rain, etc.
• the objective could be to modify the shape of flowers and/or leaves to provide more decorative forms or longer flower bearing stems as well as other desirable properties in the plants in question.
• the present inventions provides a solution to said problems by providing new methods for controlling the direction of cell growth and organ shaping in plants.
• the method and the products and means utilized in said method are as defined in the claims of the present invention.
• FIG. 1 RNA gel blot hybridisation analysis showing the tissue specificity of GEG expression.
• Figure 2. Analysis of GEG expression during corolla and carpel development.
• Figure 3. Analysis of GEG expression in carpel and corolla by in situ hybridization.
• FIG. 6 Comparison of the ray floret corollas of a non-transformed line (wt) with that of an m3 plant constitutively expressing GEG before (stage 7), during (7.5), and after (9) the opening of ray florets.
• Figure 7 Analysis of corolla length and width for four lines constitutively expressing GEG and the conn ⁇ 1 lines.
• FIG. 9 Cell lengths and widths in the same regions as described in Figure 8 of m. and m3 lines constitutively expressing GEG and a control line (wt). In plants constitutively expressing GEG, cell length was reduced but no difference in cell width could be measured.
• Figure 10 Scanning electron microscopy of the epidermis of stylar part of the carpel 300 m below the stigma. In an m ⁇ line constitutively expressing GEG, cell length was reduced and the width was increased compared to control line.
• the terms used have the meaning they generally have in the fields of conventional plant breeding, plant biochemistry and production of transgenic plants, including recombinant DNA technology as well as agriculture, horticulture and forestry. Some terms, however, are used with a somewhat deviating or broader meaning in this context. Accordingly, in order to avoid uncertainty caused by terms with unclear meaning some of the terms used in this specification and in the claims are defined in more detail below.
• GCG for "gerbera G- S-T-Mike gene” means an isolated and essentially purified cDNA sequence (Gerbera hybrida homolog for the gibberellic acid stimulated transcript 1 [G- S77], from tomato) obtainable from a library representing late stages of corolla development in Gerbera hybrida.
• GEG-like nucleic acid sequences means nucleic acid sequences homologous to said GEG-gene or cDNA.
• the "G ⁇ G-like nucleic acid sequences” are characterized by a nucleic acid sequence encoding "G ⁇ G-like gene products” having an amino acid sequence substantially homologous with the C-terminal domain (S ⁇ Q ID NO: l:) of the gene product of G ⁇ G (S ⁇ Q ID NO:2:).
• Said “G ⁇ G-like nucleic acid sequences” are further substantially similar at nucleotide level with the G ⁇ G cDNA (S ⁇ Q ID NO:3:).
• G ⁇ G-like nucleic acid sequences include the G ⁇ G-promoter S ⁇ Q ID NO:4: as well as substantially homologous promoters, which are capable of directing a foreign gene or a heterologous nucleic acid sequence in the same way as G ⁇ G is directed by its native promoter in corolla and styles.
• G ⁇ G-like nucleic acid sequences are further characterized by the capacity of spatiotemporal controlling of the plant cell growth by alternatively increasing and/or decreasing the cell growth in various directions or by directing said function.
• G ⁇ G-like nucleic acid sequences include isolated, essentially purified nucleotide sequences obtainable, for example, by differential hybridization from a group of plant genes having a high similarity with the G ⁇ G cDNA from Gerbera hybrida.
• DNA construct for altering the size and shape of plant cells or plant organs means any suitable vectors and/or DNA constructs comprising at least one GEG-like nucleic acid sequences combined with optional promoters, enhancers, signal sequences for inserting, targetting, controlling the size and shape of plants cells and plant organs.
• GEG gene product means an amino acid sequence with the deduced amino acid sequence (SEQ ID NO:2:) sharing a high similarity with previously characterized putative cell wall proteins encoded by GEG-like nucleic acid sequences.
• the "GEG-like gene products” are polypeptides characterized by having an amino acid sequence comprising amino acid sequences substantially homologous with SEQ ID NO:2:, which are further characterized by having a highly conserved C-terminal domain, with one or more invariable cysteine residues.
• the "GEG-like gene products” are further characterized by the capacity of spatiotemporal control of cell growth, which can be determined by methods disclosed in the examples.
• GEG-like gene products include in addition to said GEG-gene product, products obtainable by constitutive or induced expression of gibberellic acid-inducible genes, namely GAST1 of tomato, GIP (for gibberellin- induced gene) of petunia and the GASA (for GA-stimulated in Arabidopsis) gene family of Arabidopsis.
• GEG and GEG-like genes the expression of which can also be induced by application of exogenous gibberellic acid (GA3) plays a role in phytohormone-mediated cell expansion.
• GEG-like gene for manufacturing plants having the capability of controlling the direction and dimensions of cell growth and altering the shape of plant cells and/or plant organs.
• spatialotemporal control means that said GEG-like nucleic acid and their expression products are capable of controlling, i.e. regulating and/or adjusting the cell growth by alternatively, increasing and/or decreasing or inhibiting the cell growth in various directions, including longitudinal and/ or radial direction with adjustable, advantageous time intervals.
• substantially homologous means that the GEG-gene product have a homology of at least 40 % , preferably at least 50 % , most preferably at least 55 % at amino acid level.
• defined hybridization conditions means any hybridization conditions varying between the conditions of 58 °C, 2xSSC and 58 °C, 0.2XSSC.
• the upper limit allows the capture of genes closely related with Gerbera and the lower limit of at least 58 °C, 0.2XSSC allows the capture of the GEG-gene as such, especially when the part of the nucleic acid sequence encoding the homologous C-terminal domain is excluded.
• plant cell line means a cell line into which a GEG-like gene is inserted by per se known methods or is a cell line capable of expressing a GEG-like gene product having the capacity of controlling the direction and dimensions of cell growth, especially increasing the radial cell growth and inhibiting the longitudinal cell growth.
• transgenic plant means a plant into the cells of which at least one GEG-like gene has been introduced or integrated and which cells are capable of expressing a GEG-like gene product having the capacity of controlling the direction and dimensions of cell growth, especially increasing the radial cell growth and inhibiting the longitudinal cell growth or vice versa.
• GEG-like gene products and derivatives thereof cover all possible splice variants of the GEG-product, including truncated, complexed as well as derivatized forms of said GEG-product, which still have the capacity of spatiotemporal control of cell growth in plants.
• GEG-like gene product in its broadest aspect in the present invention, covers not only normal GEG-like molecules including their isAorms of different origin, as separate entities or in any combinations.
• the term covers all listed gene products in their active forms and in any combinations of said forms as well as fragmented, truncated, derivatized and/or complexed forms thereof, which fulfill the prerequisites defined in the previous paragraph.
• isoform refers to the different forms of the same protein, which originate from different sources, e.g. different species of plants.
• the term includes fragments, complexes and their derivatives.
• GEG-like gene products are generated e.g. by the cleavage.
• Different reactions including different enzymatic and non-enzymatic reactions, proteolytic and non-proteolytic, are capable of creating a truncated, derivatized, complexed form of the said GEG-gene product. They are incorporated in the present invention as long as they fulfill the prerequisite of capacity for spatiotemporal control of plant cell growth.
• altering means capacity of spatiotemporal control of plant cell growth, i.e. adjusting by molecular regulation the direction and dimensions of cell growth and the organ shape in plants, especially during flower development.
• the term "manufacturing plants with spatiotemporal control of cell growth” means that the plants are transgenic plants produced by incorporating (inserting) a DNA-construct or vector carrying one or more nucleic acid sequences of the present invention into a plant cell, which can be induced to express said insert by administration of gibberellic acid (GA3) or auxin or is constitutively expressing said insert.
• Said plant cells are capable of spatiotemporally directing cell growth in variable directions in order to allow reshaping of organs in plants.
• the GEG-like gene products expressed by the GEG-like nucleic acid sequences of the present invention -r e accordingly, useful for altering or adjusting the size and shape of plant cells or plant organs to its respective application.
• the GEG-like nucleic acid sequence as well as their expression products can be used for manufacturing plant cells or plants with modifiable size and/or shape by directing the plant cell growth, in various, alternatively, in longitudinal and/or radial direction to obtain the size and shape of plant cells or plant organs, which is best suited to its respective application in agriculture, horticulture and/or forestry.
• GEG-like gene/protein family shares several features that may suggest a role for G ⁇ G in regulating cell expansion.
• the "G ⁇ G-like genes and their gene products" are useful for manufacturing plants having the capability of controlling the direction and dimensions of cell growth and altering the shape of plant cells and/or plant organs.
• GEG expression in corollas and carpels coincides spatiotemporally with flower opening.
• GEG expression is temporally correlated with the cessation of longitudinal cell expansion.
• reduced corolla lengths and carpels with shortened and radially expanded stylar parts with concomitant reduction of cell expansion in these organs was observed and in styles, an increase in radial cell expansion was detected.
• the styles of carpels are fine and elongated non-photosynthetic structures.
• GEG for gerbera G- SE2-like gene
• GEG expression both spatially and temporally correlates with the opening of the corolla and with cessation of corolla elongation.
• induction of GEG expression coincides with the cessation of style elongation.
• corollas are shorter when compared to those of non-transformed lines.
• constitutive GEG expression causes shortening of the carpel, but also a concomitant radial expansion of the style.
• epidermal cells of both the ligular part of the corolla and the style are reduced in length along organ axes. Radial expansion of the epidermal cells in styles was also observed. The results suggest that said phytohormone mediated cell expansion also can be applied in agriculture and forestry in order to provide new methods and means for controlling plant growth in a desired direction.
• GEG belongs to a gene family encoding putative small cell wall proteins with a cysteine-rich domain and a putative signal peptide sequence (GASTl of tomato, GASAl-4 of Arabidopsis and GIP of petunia and RSI-1 of tomato) (Shi, L. , et al. , Plant J. 2, 153-159, 1992; Taylor, B. H. , et al. , Mol. Gen. Genet. 243, 148-157, 1994; Herzog, M. , et al. Plant Mol. Biol. 27, 743-752, 1995; Ben-Nissan, G., et al., Plant Mol.
• GEG expression was experimentally induced by a treatment with gibberellic acid (GA3), which is similar to previous reports indicating that these genes are susceptible of being regulated by gibberellic acid or auxin.
• GA3 gibberellic acid
• the results obtained indicate that GEG is part of a phytohor- mone-mediated cell expansion mechanism that functions during corolla and carpel development and that mechanism can be used for developing new methods and means for providing plant raw materials with more desired and advantageous structure for agricultural and forestral applications.
• GEG glycosylcholine
• constitutive GEG expression demonstrates that excessive GEG production is able to cause alterations in organ and cell shape during corolla and carpel development. This suggests that GEG plays a role in determining cell shape during carpel and corolla morphogenesis, thus providing functional information for the role of GEG-like genes in plants.
• constitutive GEG expression reveals a negative interrelationship between longitudinal and radial growth. As described above, this is also evident in epidermal cells. However, in corollas, no radial expansion of epidermal cells, due to constitutive GEG expression, was observed. Furthermore, in carpels, no increase in style width was observed during endogenous GEG expression stage ( Figure 4D). This would suggest that the primary role of G ⁇ G is to inhibit cell elongation. According to this hypothesis, constitutive GEG expression prematurely inhibits cell expansion in the longitudinal direction. This could lead to growth potential of the cell to be directed passively in the radial direction as seen in the epidermal cells of the style. The alternative hypothesis that G ⁇ G would promote radial and inhibit longitudinal expansion simultaneously is also possible.
• the GEG-like gene/protein family shares several features that may suggest a role for G ⁇ G in regulating cell expansion. Based on our studies of GEG expression and the fact that several members (GEG, GASTl, and RSI-1) have been isolated based on a differential screening method, we can conclude that the mRNA is relatively abundant, characteristic of a structural role for the gene product. Furthermore, the putative signal sequence and the absence of other targeting signals suggest that the gene products are secreted, possibly to the cell wall (Shi, L. , et al. , Plant J. 2, 153-159, 1992). Another characteristic feature is regulation of gene expression with phytohormones. The variability in the effective hormone indicates that the role of the genes may be downstream of various signal transduction pathways after their convergence.
• G ⁇ G like function may be generally related to establishing cell wall properties during organogenesis in plants.
• Gerbera hybrida var Terra Regina used in this research was obtained from Terra Nigra BV, Holland.
• the control and transgenic plants were grown under identical conditions (side by side) at the same time and the age of plants was same. Developmental stages of the inflorescence are described in Helariutta, Y. , et al. , Plant Mol. Biol. 22, 183-193, 1993. For all analyses, samples were collected from outermost ray florets (flowers) of the inflorescence, and each transgenic and control plant sample was harvested and treated at the same time.
• Example 2 Example 2
• Gerbera transformation was performed using Agrobacterium tumefaciens-mediated gene transfer as described previously (Elomaa, P. , et al. , Bio/technology 11, 508-511, 1993). Transformation was verified by RNA blot analysis showing GEG expression in leaves and by DNA blot analysis. The analyses have been performed on clones of the original transgenic plants (TQ).
• RNA Polyadenylated RNA (5 ⁇ g), extracted from proximal part of ray floret corollas at developmental stages from 5 to 9 (Helariutta, Y. , et al. , Plant Mol. Biol. 22, 183-193, 1993) was used to construct a cDNA library in the ZAPII vector (ZAP-cDNA synthesis kit; Stratagene, La Jolla, CA).
• plaques were plated and transferred onto replica nylon membranes, and then screened differentially with radiolabeled first-strand cDNA pools from the ray floret tube region of the proximal part and distal part of ligule (first strand cDNA synthesis kit; Amersham).
• GEG cDNA was isolated as a clone which is expressed stronger in proximal part than distal part of the ligule.
• Two independent, but similar cDNA clones were isolated, sub- cloned into pUC18 derivative, and sequenced using the AutoRead kit (Pharmacia, Uppsala, Sweden).
• the 813 bp genomic fragment containing part of GEG promoter was obtained by applying a 5' RACE like PCR amplification on genomic DNA.
• the GEG cDNA sequence and sequence of the 5' flanking region of the GEG gene have been submitted to EMBL database and the accession numbers are AJ005206 and AJ006273, re- spectively.
• RNA blots Fifteen micrograms of total RNA was loaded per lane. The amount of RNA to be loaded was measured spectrophotometrically and the equal loading was confirmed by ethidium bromide staining of rRNA bands. The electrophoresis and hybridizations were made as described in Sambrook et al. , 1989. The 259-bp long 3' fragment (of which 234 bp is from non coding region) served as the probe. Washing conditions of 0.2 SSC (1 SSC is 0.15 M NaCl and 0.015 M sodium citrate) and 0.1 % SDS, 58°C were applied in all RNA blots. In situ hybridization was carried out as described previously in Kotilainen M. , et al.
• RNA probes were transcribed from the same fragment as used in the gel blot studies under the T7 promoter in vector pSP73 (SP6/T7 transcription kit; Roche Diagnostics. Mannheim Germany).
• Corolla and carpel samples of control and transgenic plants were collected and further treated side by side at the same time. They were fixed in FAA buffer (50 % ethanol, 5 % acetic acid, and 2 % formaldehyde) overnight, and then transferred through ethanol series to 100 % ethanol, critical point dried (Balzers CPD 030 Critical Point Dryer, Bal-Tec, Liechtenstein) and coated with platinum/palladium (Agar Sputter Coater, Agar Scientific Ltd, UK).
• Specimens were mounted on aluminium stubs using graphite adhesive or tape, examined with scanning electron microscope (Zeiss Digital Scanning Microscope DSM 962, Karl Zeiss, Germany) in the Electron Microscopy Laboratory of the Institute of Biotechnology, University of Helsinki.
• Organ length and width measurements were done with vernier caliper in vivo, except for carpel width, which was measured from scaiining electron micrographs 200 ⁇ m below stigma. Cell length and width were measured by using scanning electron micrographs.
• Student s t tests and/or Rank sum tests were performed. Parametric t test was used if the normality and the equal variances of samples were confirmed (P values to reject ⁇ 0.050). Non parametric Rank sum test was used if either was not confirmed. The level of confidence is P ⁇ 0.001 in all statistically significant differences mentioned in this study.
• GEG belongs to a family of genes that are transcrip- tionally regulated by phytohormones in different plants.
• the predicted GEG protein has high sequence similarity with proteins encoded by genes whose expression is induced by gibberellic acid (GASTl of tomato, GASAl-4 of Arabidopsis, and GIP of petunia) or by auxin (RSI-1 of tomato) (Shi, L. , et al. , Plant J. 2, 153-159, 1992; Taylor, B. H., et al. , Mol. Gen. Genet. 243, 148-157, 1994; Herzog, M. , et al. Plant Mol. Biol.
• the probe (a 259-bp long 3' fragment of the GEG cDNA, 90% noncoding) recognized one or two bands at the stringency used for RNA gel blotting. This most probably indicates that the expression analysis results presented below correspond to transcription of a single locus, and that the two bands found in some digests were due to restriction length polymorphism in the heterozygous cultivar.
• the full-length GEG cDNA probe recognized more bands, suggesting that there is a small gene family of GEG-like genes in the gerbera genome (data not shown) .
• GEG mRNA is abundant in corollas and carpels
• the developmental expression pattern of GEG was studied by using RNA gel blot analyses.
• the expression of GEG is highest in floral organs, in addition a faint signal was detected in RNA from leaf blades. Strong GEG expression was observed in corolla tissue (both tube and ligule regions) and carpels, with more moderate signals in the scape (floral stem) and the receptacle (terminal enlargement of floral stem) (Figure 1).
• the temporal GEG expression pattern along the apical-basal axis of corolla made it important to analyze whether GEG expression correlates with cessation of cell elongation.
• Cell length was measured in the distal and central regions of corolla (Figure 2H, regions 7 and 5, respectively) just after stage 1 (1+) and at stage 8. At these stages, GEG mRNA is present in the distal region, but reaches the central region just prior the stage 8 ( Figure 2H).Cell length measurements revealed that cells in the distal region do not elongate, whereas in the central region, axial cell elongation takes place (Figure 5A). The cell length differences between stage 8 middle cells and other groups are statistically significant (Rank sum tests, P ⁇ 0.001). Cell width growth was detected both in distal and middle parts of corolla between stages 7+ and 8 ( Figure 5 B).Thus, GEG expression strictly correlates with the cessation of cell expansion along the apical-basal axis.
• the GEG cDNA was introduced into gerbera plants under regulation of the Cauliflower mosaic virus 35S promoter via Agrobacterium tumefaciens-me ⁇ iated transformation.
• Four constitutively GEG-expressing lines were generated, and analyses of both the length and the width of 20 outermost ray floret corollas in four transgenic plants and control plants were conducted at developmental stage 9 when corolla growth has ceased.
• Epidermal cells of the style are shorter and wider in lines constitutively expressing
• transgenic lines constitutively expressing GEG has a decrease in carpel length and an increase in carpel radius ( Figures 10, 11 A, and 11B).
• a comparison of cell length and the width of style epidermal cells between mi and 103 lines constitutively expressing GEG, and the control line revealed a change in elongation pattern. Even before endogenous expression, at stage 6, statistically significant changes of cell length and width could be detected (t test; P ⁇ 0.001).
• m and 1113 lines constitutively expressing GEG cell length was reduced and the width was increased compared to control line ( Figures 10, 11C, and 11D).
• the constitutive expression phenotypes support the view that the GEG gene product regulates cell expansion in the axial dimension during carpel development as well as during corolla development. However, in the carpel, unlike in the corolla, we observed a concomitant opposite effect in the radial dimension.
• GEG genomic 5' flanking sequence of GEG. It contains two sequence motifs that are found in the flanking regions of rice and barley -amylase genes whose expression is regulated by gibberellic acid (Huang, N. , et al.. Plant Mol. Biol. 14, 655-668, 1990; Skriver, K. , et al. , Proc. Natl. Acad. Sci. USA 88, 7266-7270, 1991. This further supports the idea that GEG expression is developmental ⁇ regulated by gibberellic acid.
• FIG. 1 RNA gel blot hybridisation analysis showing the tissue specificity of GEG expression.
• RNA gel blot probed with a 259-bp long 3' fragment of the GEG cDNA (90 % noncoding). Fifteen micrograms of total RNA was loaded per lane and equal loading was confirmed by ethidium bromide staining. Organs covering several developmental stages were examined. Scape, floral stem; receptacle, terminal enlargement of floral stem.
• (A) The developmental stage is 1, (B) 3, (C) 5, (D) 7, (E) 7.5, and (F) 8.
• (G) The expression of GEG in carpel and corolla correlates with opening of both individual ray florets and the whole inflorescence. Spatial partition of ray floret corolla. Regions are indicated above the gel. The onset of expression occurs from both ends of the corolla (stage 7, regions 2 and 7) and just as the corolla has opened. Both expression domains meet at the middle of the ligule (stage 8, region 5). Region 1 is the tubular part of ray floret corolla (tube); region from 3 (proximal region) to 7 (distal region) represent the ligular part of the corolla. GDFR1 is used as a loading control. The developmental stages are the same as presented in (A) to (F).
• FIG. 3 Analysis of GEG expression in carpel and corolla by in situ hybridization. In both organs (stage 7.5), GEG expression is seen in epidermal and parenchymatic cells as white silver grains. In situ analysis were carried out using the 35s_c ⁇ p-i a beled antisense and sense (control, data not shown) RNA probes. The probes were transcribed from the same 3' fragment of GEG cDNA as used in RNA gel blot analysis.
• Timing of different developmental stages was measured by following the development more than 50 inflorescences under our standard greenhouse conditions.
• Carpel cell length (E) of 72 epidermal cells of each time point was measured. Cell lengths of 18 epidermal cells 200 - 400 ⁇ m below the stigma of each carpel were determined, and the average cell length of four carpels was measured.
• GEG is expressed in proximal part at both stages, whereas GEG expression reaches the central region just prior the stage 8 (see Figure 2H).
• FIG. 6 Comparison of the ray floret corollas of a non transformed line (wt) with that of an ⁇ -3 plant constitutively expressing GEG before (stage 7), during (7.5), and after (9) the opening of ray florets.
• Figure 7 Analysis of corolla length and width for four lines constitutively expressing GEG and the control lines.
• Corolla length (mm). Length of the outermost ray floret corollas of wild-type, constitutively GEG expressing lines (mj , ⁇ -3, m2, and 015), and two GEG antisense lines with no ) or a modest (-9, 80% remaining) decline of GEG expression. All four lines constitutively expressing GEG have statistically shorter corollas compared to the control lines.
• FIG. 8 Analysis of the effects of constitutive GEG expression on corolla epidermal cells.
• the epidermal cells are organized into longitudinal files running along the apical-basal axis of corolla.
• (B) Scanning electron microscopy of the adaxial (upper) side of proximal part of ray floret corolla of a non transformed control line (stage 8).
• the area in (B) is marked with a white box in (A).
• One of the epidermal cells is highlighted.
• FIG. 9 Cell lengths and widths in the same regions as described in Figure 8 of mi and m3 lines constitutively expressing GEG and a control line (wt). In plants constitutively expressing GEG, cell length was reduced but no difference in cell width could be measured.
• transgenic lines mi and m3 together with a non transformed control line were collected (stage 8), and cell length and width were measured at the region marked with a white box in the Figure 8A.
• transverse lines were drawn on micrographs, and cells were chosen at intervals of 1 cm for length measurements.
• Cell length of about 200 cells were measured of mi, 1113, and a control line.
• Cell width was measured by counting cell numbers on 30 - 36 of 570 ⁇ m transverse lines and counting the average cell width of each line (approximately 40 cells per line).
• Figure 10 Scanning electron microscopy of the epidermis of stylar part of the carpel 300 m below the stigma. In an mi line constitutively expressing GEG, cell length was reduced and the width was increased compared to control line.
• the epidermal cells of constitutively GEG expressing lines are wider when compared to a control line.
• the scape (floral stem) was cut 5 cm below inflorescence.

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• 1999
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