Journal of Experimental Botany, Vol. 53, No. 366, pp. 51-59,
January 1, 2002
© 2002 Oxford University Press
Original Papers |
Regulation of expression of two novel flower-specific genes from tomato (Solanum lycopersicum) by gibberellin
Department of Experimental Botany, Research Group Molecular Plant Physiology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
Received 22 March 2001; Accepted 8 August 2001
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Abstract |
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Two flower-specific cDNAs have been isolated after differential screening of an anther cDNA library. This library was constructed 48 h after GA3 treatment of buds of the GA-deficient gib-1 mutant of tomato. Northern blot analysis during flower development in tomato demonstrated that the expression of both genes is regulated by gibberellins (GAs). Application of GA3 to developmentally arrested gib-1 flower buds induced new expression of tgas100 mRNA 48 h post-treatment, while an increased accumulation of tgas105 mRNA was found after 8 h. In situ analyses showed the spatial distribution of the expression of both genes within the tomato flower. One of the deduced polypeptides (TGAS105) displays similarities to cysteine-rich extensin-like proteins, while the other (TGAS100) shows significant homology with a stamen-specific gene of Antirrhinum majus. Based on the deduced protein sequences, the possible function of the encoded proteins is discussed.
Key words: Extensin-like, flower-specific gene expression, gibberellin, Solanum lycopersicum.
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Introduction |
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Plant growth and development is a very complex process in which endogenous and exogenous factors are involved in inducing and regulating different developmental processes. Gibberellins (GAs) are endogenous plant growth regulators that are involved in the regulation of tuberization, seed germination, extension growth, and flowering (for reviews, see Kende and Zeevaart, 1997
; Vreugdenhil and Sergeeva, 1999; Evans, 1999; Thornton et al., 1999; Blazquez and Weigel, 2000). Although the molecular mechanisms by which plants respond to gibberellins are still largely unknown, several components of the signal transduction pathway have been identified. One component of the pathway, SPINDLY, is believed to be an O-GlcNAc transferase that post-translationally modifies cytosolic and nuclear proteins (Thornton et al., 1999). The classification of GA-response mutants in two categories, suggests that there are at least some common elements in the different responses of plant cells to GA. The first category consists of dwarf mutants that have a reduced response to both endogenous and applied GA (GA-insensitive mutants). The second category, slender mutants, exhibit phenotypes suggestive of an over-response to GA.
Specific progress towards understanding the mechanism of GA action has been made for the aleurone layer of germinating barley and wheat, where GAs induce the synthesis and secretion of a number of hydrolytic enzymes (Cercos et al., 1999).
Gibberellins are essential for the development of fertile flowers in tomato, and may also be required immediately after fertilization. In order to study the molecular mechanisms of GA in flower development, their role in the promotion of anther development has been investigated in tomato (Solanum lycopersicum). As a model system, the GA-deficient gib-1 tomato mutant was used. The mutant is deficient in GAs because its ability to convert geranylgeranyl pyrophosphate to copalyl pyrophosphate is reduced (Bensen and Zeevaart, 1990). The phenotype of this mutant, which includes dwarfism, failure to germinate and failure to flower normally, is reversed by exogenously applied GAs (Koornneef et al., 1990; Jacobsen and Olszewski, 1991). Flower initiation in the mutant is normal, but anther development is arrested just before the initiation of meiosis (Jacobsen and Olszewski, 1991). Developmentally arrested anthers contain pollen mother cells, which are in the G1 phase of the premeiotic interphase, and outer and inner tapetum cells, which are at the uninucleate and binucleate stages, respectively. The development of anthers arrests when the flower bud is 2.5 mm in length, and remains arrested, but the anthers are responsive to treatment with gibberellic acid (GA3) until the bud reaches a length of 3.7 mm (Jacobsen and Olszewski, 1991).
An approach to understanding GA action is the isolation of GA-regulated genes and to use them as molecular markers for the response of anthers to GA. These genes may also be used as tools for the identification of other factors involved in their regulation, for instance by reverse genetics. GA-regulated genes have been isolated from the aleurone layer of germinating cereal seeds, vegetative shoot tissue and the flower (reviewed in Hooley, 1994; Huttly and Phillips, 1995; Cercos et al., 1999). Also in anthers of developmentally arrested gib-1 flowers, the application of GA causes specific changes in gene expression (Jacobsen et al., 1994), as is further demonstrated in this paper.
The aim was to identify GA3-stimulated genes after differential screening of a tomato gib-1 anther cDNA library. Two cDNA clones, tgas100 and tgas105, were shown to be flower-specific and were analysed further. The temporal and tissue-specific expression of the corresponding genes and the localization of the transcripts are described. Their putative function during early flower development in tomato will be discussed.
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Materials and methods |
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Plant material
Seeds of the tomato cultivar Moneymaker, containing the gib-1 mutation, were kindly provided by M Koornneef, Department of Genetics, Agricultural University, Wageningen, The Netherlands. After spraying the plants with 1x10-3 M GA3 the seeds of the mutant were germinated in Petri dishes and then transferred to soil. The wild-type tomato (Solanum lycopersicum cv. Moneymaker) and the gib-1 mutant were grown on soil in the greenhouse at 18–22 °C with supplemental light (16 h photoperiod, 400 W high-pressure sodium lamps (Son-T)).
Hormone treatment
2 µl 7x10-5 M gibberellic acid (GA3) or dilutions of the GA3 stock solution was pipetted onto developmentally arrested gib-1 flower buds (50 ng GA3 bud-1) of about 2.5-month-old plants. For measuring the effect of abscisic acid (ABA) or benzylaminopurine (BAP) on the GA3 treatment, an additional 4 µl 5x10-4 M solution with these hormones was applied to the buds.
cDNA library construction and differential screening
The gib-1 anthers of flower buds with a length between 2.5 and 3.7 mm were harvested 48 h after the application of 50 ng GA3 bud-1. Total RNA was isolated from plant tissues using the method developed earlier (Van Eldik et al., 1995). cDNAs were synthesized from total RNA, using a Uni-ZAP XR cDNA synthesis kit (Stratagene), according to the protocols of the manufacturer. The library was packaged using Gigapack II gold packaging extracts (Stratagene). Differential screening of the cDNA library was performed on nitrocellulose with 32P-labelled single-stranded cDNA probes with a specific activity of approximately 5x108 cpm µg-1. The cDNA probes were prepared from total RNA of gib-1 anthers (Neuteboom et al., 1999), 48 h after treatment with either GA3 or H2O (Sambrook et al., 1989). Excision of the positive cDNA clones from lambda ZAP II to yield the pBluescript II SK(-) vector in XL-1 Blue E.coli cells (Stratagene) was carried out with the ExAssist/SOLR in vivo excision system (Stratagene).
DNA manipulations and sequence analysis
Plasmid DNA was prepared as described previously (Sambrook et al., 1989). Radioactively labelled probes were made from isolated cDNA fragments using the random-primer labelling system (Feinburg and Vogelstein, 1984). Sequencing of both DNA strands of the cDNA clones was carried out by using the PRISM Sequencing kit (Perkin Elmer). Computer analysis of both nucleotide and deduced protein sequence was performed using the University of Wisconsin Computer Group programs (Devereux et al., 1984).
Northern and Southern blot analysis
Equal amounts (10 µg) of total RNA from different tissues or treatments were electrophoretically separated on 1% MOPS/formaldehyde–agarose gels and transferred to Hybond-N (Amersham) (Sambrook et al., 1989). Membranes were hybridized under standard conditions at 55 °C with radiolabelled probe of TGAS118 as described before (Van den Heuvel et al., 1999). Equal RNA loading was monitored on the agarose gel under UV light or by hybridizing the Northern blot with a radiolabelled 28 S ribosomal RNA cDNA fragment isolated from Nicotiana tabacum cv. Petit Havana. The membrane filters were exposed to Valva HPX44 or the more sensitive Kodak X-omat AR X-ray films (normal exposure 2–4 d, longer exposure 2–3 weeks).
Genomic DNA (40 µg, extracted from young leaves according to Bernatzky and Tanksley, 1986), was digested with EcoRI or HindIII and electrophoretically fractionated on 1% agarose gels before transfer to Hybond-N as described previously (Sambrook et al., 1989). After prehybridization of the blots at 62 °C, as described for Northern blot hybridization, hybridization was carried out for 20 h at 62 °C with radiolabelled tgas100 and tgas105 probes.
In situ hybridization
The wild-type flower buds of different lengths were collected and fixed in PAGA buffer (3% paraformaldehyde, 0.1% glutaraldehyde, 0.1 M NaCl, 10 mM Na2HPO4, and 10 mM NaH2PO4, pH 6.8). After dehydration, the buds were embedded in Paraplast Plus (Sigma), 10 µm thick sections were cut and mounted on slides (Angerer et al., 1987). The mounted slides were deparaffinized, hydrated and (pre)hybridized (Cox and Goldberg, 1988; de Almeida-Engler et al., 1994). Both antisense and sense probes of tgas100 and tgas105 cDNA cloned in pBluescript II SK(-) were prepared by using (T3/T7) DIG RNA Labelling Kit (Boehringer Mannheim). A probe concentration of 40 ng ml-1 was used for overnight hybridization at 42 °C. Hybridization and performance of the detection reaction were carried out according to the manufacturer's instructions (Boehringer Mannheim).
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Results |
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Tissue-specificity and developmental regulation of tgas100 and tgas105 expression
Differential screening of a tomato gib-1 anther cDNA library, constructed 48 h after GA3 treatment of developmentally arrested gib-1 buds, allowed the isolation and characterization of cDNAs derived from GA3-upregulated mRNAs. Several GA3-stimulated cDNAs were found by using this protocol, of which tgas100 and tgas105 appeared to be flower-specific and were analysed further.
The tissue-specific expression pattern of the tgas100 and tgas105 gene was determined by Northern blot analysis using total RNA, which was extracted from various tissues of the wild-type tomato plant (Fig. 1). The tgas100 cDNA hybridized to a single mRNA transcript of 0.61 kb present in anthers and at a lower level in petals. Hybridization with tgas105 cDNA as a probe resulted in a strong signal corresponding to a mRNA of 0.61 kb that was detected in petals, anthers and pistils (Fig. 1). Both transcripts were not detected in any other tissue even after extended exposure of more than 2 weeks.
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To detect tgas100 and tgas105 mRNAs during flower development, Northern blot analysis was performed with RNA extracted from wild-type buds and untreated gib-1, which was probed with radiolabelled tgas100 and tgas105 cDNA. The tgas100 mRNA was abundantly present in wild-type buds with a length of 4–8 mm and was not detected in mutant buds (Fig. 2
). The tgas105 mRNA was already present in 2 mm wild-type buds, reached a maximum in 5 mm buds and decreased thereafter. Only a weak signal was detected in mature flowers. A cross-hybridizing transcript of about 0.9 kb appeared in 8 mm buds until mature flowers (Fig. 2). In the gib-1 mutant the tgas105 mRNA was detected in 2 mm buds, but diminished during flower development (Fig. 2). Without GA treatment the tgas105 transcript was present until a bud reached a length of 6 mm after which stage the buds eventually aborted.
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Spatial expression of tgas100 and tgas105
The expression of tgas100 and tgas105 in flower buds was localized by in situ hybridization carried out on transverse sections of wild-type buds. Figure 3 shows representative results of the hybridizations. As a control, transverse sections of 5 mm buds were hybridized with a sense probe of tgas100 (Fig. 3A). Although the nuclei of the pollen mother cells showed some non-specific staining, no hybridizing signal was observed. Hybridization of an antisense probe of tgas100 resulted in a strong hybridization signal which was detected in the tapetum of the anthers (Fig. 3B). Using an antisense probe of tgas105, in the anthers of 4 mm flower buds a strong signal was detected in the tapetum and pollen mother cells (Fig. 3D). Tgas105 mRNA was also abundantly present in epidermal and parenchyma cells at the edge of the petals, and in transmitting and vascular tissue of the pistil (not shown). In the ovary tgas105 mRNA was present in the ovules (Fig. 3E). As shown in Fig. 3C, hybridization with a sense probe of tgas105 showed no hybridization in any tissue examined.
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Hormonal-regulated expression of tgas100 and tgas105 mRNAs
In order to determine the kinetics of the GA3-stimulated accumulation of tgas100 and tgas105 mRNAs in gib-1 flower buds, a time-course experiment was performed (Fig. 4). Northern blot analysis showed that the abundance of tgas105 mRNA, as shown in untreated buds, enhanced 8 h after treatment, while tgas100 mRNA was not detectable in gib-1 buds until 48 h after treatment. In wild-type buds treatment with GA3 (+) resulted in an increase of the mRNA of both genes after 48 h as compared to the control (-) (Fig. 4, left panel).
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To determine the dose-dependency of the expression, developmentally arrested gib-1 buds were treated with a range of GA3 concentrations per bud for 72 h (Fig. 5
). This time scale (72 h) was chosen to circumvent the delay in the enhancement effect of the GA, as shown for the expression of tgas100 (Fig. 4
). Northern blot analysis revealed that at 72 h post-treatment, expression of tgas100 mRNA was weakly induced after application of 5x10-10 g GA3 bud-1, while a stronger signal was found with 5x10-8 g GA3 bud-1. The tgas105 transcript, weakly present in the untreated buds, showed an increased accumulation with 5x10-10 g GA3 bud-1 (lane 2).
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The effect of the GA3 application in combination with ABA or BAP was tested on the accumulation of tgas100 and tgas105 mRNA. As shown in Fig. 6
, ABA acted antagonistically on the GA3-stimulated tgas105 enhancement, while no additional effect of BAP was observed 48 h post-treatment. An additional stimulatory effect of both ABA and BAP was observed on the tgas100 expression 48 h after application (Fig. 6). Since it was shown earlier that different genes react differently to ABA (e.g. tgas118, Van den Heuvel et al., 2001) the reproducibility of the differences in response of tgas100 and tgas105 on ABA and BAP was confirmed.
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Characterization of GA3-upregulated cDNAs tgas100 and tgas105
The recombinant tgas100 clone of 543 bp contained a full-length sequence, while rescreening with the 486 bp non-full length tgas105 cDNA resulted in the isolation of a 602 bp long full-length clone for tgas105. In this respect, full-length means from the start codon to the polyA tail. The polyadenylated tgas100 cDNA (with EMBL accession number AJ133599) showed an open reading frame that encodes a 104 amino acid long protein with a calculated molecular mass of about 11.9 kDa. The encoded protein sequence contains a hydrophobic putative signal peptide, but the putative cleavage site located between amino acids 20 and 21 does not confirm to the (-3; -1) rule (von Heijne, 1986). However, the hydrophobic 26 N-terminal amino acids have the characteristics of a potential transmembrane helix and therefore the TGAS100 polypeptide could be an integral membrane protein. Following the hydrophobic region there is a domain abundant with cysteine (12) and charged (28) amino acid residues. The cysteines are located in two C(X3)C(X3)C motifs and two C(X3)C(X4–6)C motifs, where X can be any residue. A similar pattern of cysteine motifs is found in TAP1, a stamen-specific gene of Antirrhinum majus (Nacken et al., 1991). The putative TGAS100 protein sequence shows 34% identity with TAP1 and, including conservative substitutions, both sequences share 62% similarity. Figure 7 shows the alignment of the TGAS100 peptide with the 107 amino acids of TAP1. Although the TAP1 protein contains a putative signal peptide sequence, protein analysis of the entire protein revealed that the hydropathy plot (Kyte and Doolittle, 1982) of both is similar (plot not shown).
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The 602 bp long tgas105 cDNA clone (with EMBL accession number AJ133600) has an open reading frame corresponding to a protein of 137 amino acid residues and a predicted molecular mass of about 15.3 kDa. The N terminus contains the features of a signal peptide with a processing site located between amino acids 23 and 24, which confirms to the (-3; -1) rule (von Heijne, 1986
). Another feature of the deduced protein is the presence of a short proline rich domain (residues 41–64), where the proline residues are distributed in two S–P3–5 motifs and four X–P doublets (with X being serine, threonine, or tryptophan). The content of proline residues is 11.7% of the entire protein, and 50% within the proline domain. Furthermore the TGAS105 protein is relatively cysteine rich (8%), with cysteines mainly distributed in the C-terminal region (residues 65–137). Similar features are found in the cysteine-rich extensin-like proteins (CELPs) of tobacco (Wu et al., 1993) which share homology (59% identity) with the TGAS105 protein sequence. In addition, the TGAS105 peptide revealed homology with two partial cDNAs for class I extensin-like proteins of tobacco (41.5% identity) (Goldman et al., 1992), and 29.5% identity with a partial cDNA for an extensin-like protein of Solanum tuberosum (EMBL accession number AJ003220). The deduced TGAS105 protein is the smallest of the reported extensin-like proteins. The difference in the overall length can be due to the presence of a shorter proline-rich domain compared to the CELPs (Fig. 8). Most significant homology is found in the C-terminal region, where the distribution of the cysteines is well conserved (Fig. 8). Computer analysis (Prosite search, Bairoch et al., 1997) revealed that the protein sequence contains one potential N-glycosylation site and four casein kinase II phosphorylation sites.
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Genomic organization of the tgas100 and tgas105 gene family
The genomic organization of the tgas100 and tgas105 gene was determined by Southern analysis. Genomic DNA isolated from Solanum lycopersicum cv. Moneymaker and digested with EcoRI or Hind III was probed with tgas100 and tgas105 cDNA in different experiments. As shown in Fig. 9, one hybridizing fragment, which was detected with the tgas100 probe suggests that tgas100 is a single copy gene. A single hybridizing band was also determined in Petunia hybrida, Solanum tuberosum and Nicotiana tabacum cv. Petit Havana (data not shown). Hybridization under standard conditions with tgas105 also revealed, in both lanes, one strong hybridizing band (Fig. 9), indicating that tgas105 is also a single copy gene.
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Discussion |
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A better understanding of the GA action on early flower development in tomato can be obtained with the identification and isolation of genes that are up-regulated by this hormone. Earlier work has shown that a developmental arrest occurs in the anthers of the gib-1 tomato mutant prior to meiosis (Jacobsen and Olszewski, 1991
). Treatment of the GA-deficient mutant with GA3 causes restoration of flower development accompanied by changes in the abundance of a number of translatable mRNAs, of which one population showed increased accumulation at 8 h post-treatment, while another population was newly expressed after 48 h (Jacobsen and Shi, 1994). Two flower-specific cDNAs, of which GA3 regulates the abundance, were isolated and characterized, one from each population.
tgas105
One of the first changes observed in gib-1 buds, whose development has been rescued, is the increase in abundance of tgas105 mRNA 8 h post-treatment. The putative TGAS105 protein shares homology with tobacco extensin-like proteins (Goldman et al., 1992; Wu et al., 1993). With the tobacco extensin-like proteins, the encoded TGAS105 protein differs from the extensins by a low tyrosine content, a lower copy number of the Ser-Pro4 motif, and a high cysteine content. Some of the proline residues in the proline-rich domain are probably hydroxylated and these hydroxyprolines may provide additional sites for glycosylation (Kieliszewski and Lamport, 1994). However, it remains to be determined whether the presence of a putative signal peptide means that the TGAS105 protein is secreted, like the CELP proteins which are localized mainly at the cell walls (Wu et al., 1993).
The tgas105 RNA is expressed in a tissue-specific manner in petals, anthers, pistil, and ovary. The development of these organs is known to be affected by GA-deficiency in the gib-1 (Jacobsen and Olszewski, 1991) and gib-2 (Nester and Zeevaart, 1988) mutants of tomato. Although the function of the protein remains to be determined, the cell-specific and developmentally regulated expression suggests that the TGAS105 protein is functionally important in cell walls in particular. An attractive model is that TGAS105 is involved in the control of cell wall extensibility, which is one factor that determines the growth of plant cells. The elongation of cells and the polarity of growth are known to be regulated by GAs (for review, see Kende and Zeevaart, 1997) and the cellular basis of the gib-1 phenotype might be both a reduced cell elongation and cell division (Koornneef et al., 1990) due to the absence of GA. Playing a role in cell wall extensibility the protein does not necessarily have a solely structural role, but may function in cell to cell interaction.
The hormone ABA counters the GA3-enhanced tgas105 mRNA accumulation. In tomato, exogenous application of ABA, an antagonist of GA3 action in many systems, induces male sterility by affecting flower development (Chandra Sekhar and Sawhney, 1991). It has been suggested that for deepwater rice the balance between the ABA and GA3 contents determines the growth rate of internodes in response to submergence (Hoffmann-Benning and Kende, 1992). The growth response is mediated by ethylene and GA3, but can be inhibited by ABA treatment. Furthermore, ethylene acts by partly increasing the responsiveness to GA3 and reducing the endogenous ABA levels. The balance between ABA and GA3 is also changed in the male sterile sl-2 tomato mutant, where GA3 levels are reduced and ABA content is increased compared to wild-type plants (Sawhney and Shukla, 1994). Fertility can be regained in the sl-2 mutant after GA treatment. If ABA affects flower development by affecting cell growth, this may support the hypothesis that the TGAS105 protein plays a role in the control of cell wall extensibility.
tgas100
At developmental arrest the gib-1 anthers contain pollen mother cells that are in the G1 phase of the premeiotic interphase, and outer and inner tapetum cells, which are at the uninucleate and binucleate stages, respectively (Jacobsen and Olszewski, 1991). In the gib-2 mutant it has been shown that meiosis does not occur and that degeneration of the tapetal layer is followed by degeneration of the pollen mother cells unless the flower bud is treated with GA3 (Nester and Zeevaart, 1988). The tgas100 gene, predominantly expressed in the tapetum, is not detectable until 48 h after GA3 treatment. This late GA3-responsiveness of tgas100 suggests that the gene is regulated more indirectly by GA3 and expressed as result of the induction of normal flower development in the gib-1 mutant. The timing of the GA3-induced tgas100 expression correlates with the onset of its expression in 4 mm wild-type flower buds as the buds grow 0.5–0.7 mm d-1 after treatment with GA3.
In tomato, low light conditions induce flower abortion at the pollen mother cell stage which can be prevented by the application of GA3 plus BAP (Kinet, 1987). BAP stimulates growth of the inflorescence but it does not induce complete development to anthesis unless GA3 is applied. In the gib-1 mutant complete flower development through anthesis is obtained only with GA3 and not with other plant growth regulators (Jacobsen and Olszewski, 1991). Although GA3 is required for obtaining expression of tgas100, an additional enhancement of the accumulation is found when arrested gib-1 buds are treated with GA3 plus BAP. The antagonistic action of ABA on the GA3-induced tgas105 expression is not observed for tgas100.
The tapetum, a metabolically highly active tissue, fulfils several functions that support pollen formation. The expression of tgas100 correlates with tapetum development and degeneration. Around meiosis, when the buds are 4 mm in length, the tgas100 mRNA starts to accumulate when tapetum undergoes endomitosis whereas expression declines in buds of 9 mm when the tapetum has almost degenerated. The tgas100 clone shares most similarity with a tapetum-specific tap1 gene of Antirrhinum majus. The TGAS100 protein is most likely to be an integral membrane protein. Although, the gene is predominantly expressed in the tapetum, the protein may not fulfil a solely tapetal function, because the gene is also expressed in petals.
In conclusion, the two isolated cDNAs are specifically expressed in tomato flowers and GA is required for the regulation of their developmental expression. Analysis of the GA-deficient gib-1 mutant has shown that both cDNAs belong to different classes of GA-upregulated genes with respect to the kinetics of their accumulation after treatment. Furthermore, the spatial localization and the sensitivity to GA treatment make these two single or low copy genes useful as potential markers for studying the mode of action of GA.
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Acknowledgments |
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The authors thank Dr SE Jacobsen and Dr NE Olszewski for providing the gib-1 anther cDNA library and G van der Weerden and T Geurtz for culturing the plants. The investigations were supported by the Life Sciences Foundation (SLW), which is subsidized by the Netherlands Organization for Scientific Research (NWO). The nucleotide sequence data are reported in the EMBL Nucleotide Sequence Database under the accession numbers AJ133599 (tgas100) and AJ133600 (tgas105).
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Notes |
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1 To whom correspondence should be addressed. Fax: +31243652787. E-mail: wullems{at}sci.kun.nl
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
de Almeida-Engler J, van Montagu M, Engler G. 1994. Hybridization in situ of whole-mount messenger RNA in plants. Plant Molecular Biology Reporter 12, 321–331.
Angerer LM, Cox KH, Angerer RC. 1987. Demonstration of tissue-specific gene expression by in situ hybridization. In: Berger S, Kimmel A, eds. Methods in enzymology, guide to molecular cloning techniques. London, UK: Acadamic Press, 649–661.
Bairoch A, Bucher P, Hofmann K. 1997. The PROSITE database, its status in 1997. Nucleic Acids Research 25, 217–221.
Bensen RJ, Zeevaart JAD. 1990a. Comparison of ent-kaurene synthetase A and B activities in cell-free extracts from young tomato fruits of wild-type and gib-1, gib-2, and gib-3 tomato plants. Journal of Plant Growth Regulation 9, 237–242.
Bernatzky R, Tanksley SD. 1986. Genetics of actin related sequences in tomato. Theorethical and Applied Genetics 72, 314–321.
Blazquez MA, Weigel D. 2000. Intergration of inductive signals in Arabidopsis. Nature 404, 889–892.[Medline]
Cercos M, Gomez-Cadenas A, Ho THD. 1999. Hormonal regulation of a proteinase gene, EPB-1, in barley aleurone layers: cis- and trans-acting elements involved in the co-ordinated gene expression regulated by gibberellins and abscisic acid. The Plant Journal 19, 107–118.[Web of Science][Medline]
Chandra Sekhar KN, Sawhney VK. 1991. Role of ABA in stamen and pistil development in normal and solanifolia mutant of tomato (Lycopersicon esculentum). Sexual Plant Reproduction 4, 279–283.
Cox KH, Goldberg RB. 1988. Analysis of plant gene expression. In: Shaw CH, ed. Plant molecular biology: a practical approach. Oxford, UK: IRL Press.
Devereux J, Haeberli P, Smithies O. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12, 387–395.
Evans LT. 1999. Gibberellins and flowering in long day plants, with special reference to Lolium temulentum. Australian Journal of Plant Physiology 26, 1–8.
Feinburg AP, Vogelstein B. 1984. A technique for radiolabelling DNA fragments to high specific activity. Analytical Biochemistry 137, 266–267.[Web of Science][Medline]
Goldman MH, Pezzotti M, Seurinck J, Mariani C. 1992. Developmental expression of tobacco pistil-specific genes encoding novel extensin-like proteins. The Plant Cell 4, 1041–1051.
Hoffmann-Benning S, Kende H. 1992. The role of abscisic acid and gibberellin in the regulation of growth in rice. Plant Physiology 99, 1156–1161.
Hooley R. 1994. Gibberellins: perception, transduction and responses. Plant Molecular Biology 26, 1529–1555.[Web of Science][Medline]
Huttly AK, Phillips AL. 1995. Gibberellin-regulated plant genes. Physiologia Plantarum 95, 310–317.
Jacobsen SE, Olszewski NE. 1991. Characterization of the arrest in anther development associated with gibberellin deficiency of the gib-1 mutant. Plant Physiology 97, 409–414.
Jacobsen SE, Shi L, Xin Z, Olszewski NE. 1994. Gibberellin-induced changes in the translatable mRNA populations of stamens and shoots of gibberellin-deficient tomato. Planta 192, 372–378.[Web of Science][Medline]
Kende H, Zeevaart JAD. 1997. The five ‘classical’ plant hormones. The Plant Cell 9, 1197–1210.[Web of Science][Medline]
Kieliszewski MJ, Lamport DTA. 1994. Extensin: repetitive motifs, functional sites, post-translational codes and phylogeny. The Plant Journal 5, 157–172.[Web of Science][Medline]
Kinet JM. 1987. Inflorescence development in tomato: control by light, growth regulators and apical dominance. Plant Physiology 6, 121–127.
Koornneef M, Bosma TDG, Hanhart CJ, Van der Veen JH, Zeevaart JAD. 1990. The isolation and characterization of gibberellin-deficient mutants in tomato. Theoretical and Applied Genetics 80, 852–857.
Kyte J, Doolittle RF. 1982. A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157, 105–137.[Web of Science][Medline]
Nacken WKF, Huijser P, Beltran J-P, Saedler H, Sommer H. 1991. Molecular characterization of two stamen-specific genes, tap1 and fil1, that are expressed in the wild type, but not in the deficiens mutant of Antirrhinum majus. Molecular and General Genetics 229, 129–136.
Nester JE, Zeevaart JAD. 1988. Flower development in normal tomato and a gibberellin-deficient (ga-2) mutant. American Journal of Botany 75, 45–55.
Neuteboom LW, Ng JMY, Kuyper M, Clijsdale OR, Hooykaas PJJ, Van der Zaal BJ. 1999. Isolation and characterization of cDNA clones corresponding with mRNAs that accumulate during auxin induced lateral root formation. Plant Molecular Biology 39, 273–287.[Web of Science][Medline]
Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning—a laboratory manual, 2nd edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Sawhney VK, Shukla A. 1994. Male sterility in flowering plants: are plant growth substances involved? American Journal of Botany 81, 1640–1647.
Thornton TM, Swain SM, Olszewski NE. 1999. Gibberellin signal transduction presents ... the SPY who O-GlcNAc'd me. Trends in Plant Science 4, 424–428.[Web of Science][Medline]
Van den Heuvel KJPT, Van Esch RJ, Barendse GWM, Wullems GJ. 1999. Isolation and molecular characterization of gibberellin-regulated H1 and H2B histone cDNAs in the leaf of the gibberellin-deficient tomato. Plant Molecular Biology 39, 883–890.[Web of Science][Medline]
Van den Heuvel KJPT, Barendse GWM, Wullems GJ. 2001. The expression of tgas118, encoding a defensin in Solanum lycopersicon, is regulated by gibberellin. Journal of Experimental Botany 52, 1–10.
Van Eldik GJ, Vriezen WH, Wingens M, Ruiter RK, Van Herpen MMA, Schrauwen JAM, Wullems GJ. 1995. A pistil-specific gene of Solanum tuberosum is predominantly expressed in the stylar cortex. Sexual Plant Reproduction 8, 173–179.
Von Heijne G. 1986. A new method for predicting signal sequence cleavage sites. Nucleic Acids Research 14, 4683–4690.
Vreugdenhil D, Sergeeva LI. 1999. Gibberellins and tuberization in potato. Potato Research 42, 471–481.
Wu H-M, Zou J, May B, Gu Q, Cheung AY. 1993. A tobacco gene family for flower cell wall proteins with a proline-rich domain and a cysteine-rich domain. Proceedings of the National Academy of Sciences, USA 90, 6829–6833.
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