Journal of Experimental Botany, Vol. 52, No. 360, pp. 1427-1436,
July 1, 2001
© 2001 Oxford University Press
Original Papers |
The expression of tgas118, encoding a defensin in Lycopersicon esculentum, is regulated by gibberellin1
Department of Molecular Plant Physiology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, Netherlands
Received 12 October 2000; Accepted 7 March 2001
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Abstract |
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A flower specific cDNA, tgas118, has been isolated after differential screening of a gib-1 anther cDNA library of Lycopersicon esculentum. The corresponding mRNA was present in all tissues analysed. Northern blot analysis revealed that in wild-type tomato the gene was predominantly expressed throughout flower development, while in the gibberellin (GA)-deficient mutant of tomato (gib-1) the abundance declined. Treatment of the mutant with GA resulted in an accumulation of the tgas118 mRNA within hours in leaf and bud tissues. In the leaf, GA1, GA3 and GA9 were effective in enhancing the expression while GA4 was not. In addition to GA, wounding and dehydration also increased the accumulation of tgas118 mRNA in leaf tissue. In situ hybridization showed that application of 50 ng GA3 bud-1 induced a similar spatial expression of the tgas118 mRNA in gib-1 buds 24 h post treatment to that found in wild-type flower buds. The deduced TGAS118 protein displays up to 77% similarity with defensins and as its expression is up-regulated by stimuli such as wounding it is proposed that it may play a role in protection against pathogens.
Key words: Gene expression, gibberellins, defensins, plant defence, Lycopersicon esculentum.
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Introduction |
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Gibberellins (GAs) are endogenous plant growth regulators (PGRs) involved in the regulation of many aspects of plant growth and development such as 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). The multiple roles of GAs in plant development have been investigated using a number of approaches, including the application of specific GAs and GA biosynthesis inhibitors to whole plants, tissues and plant cells and the identification of mutants defective in GA biosynthesis or responses to this PGR. The recent cloning of genes encoding GA biosynthetic enzymes and signal transduction intermediates has resulted in an increase in knowledge of the metabolism and function of this PGR (Cercos et al. 1999).
The response mutants, which have an altered response to biologically active GAs, consist of two main classes. These comprise firstly dwarf or semi-dwarf GA-insensitive mutants which do not properly respond to GAs and, secondly, constitutive GA responders (slender mutants) that have a phenotype similar to a wild-type plant treated repeatedly with GA (Hooley, 1994).
A complementary approach to understand GA action is to isolate genes that are regulated by GAs. Such 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). They can be used as molecular markers for response to GA and as tools to isolate the molecules that are responsible for this regulation. Both approaches contribute to a better understanding of the signal transduction pathway from GA perception to gene activation.
The gib-1 mutant, a GA-deficient mutant of tomato (Lycopersicon esculentum), shows a phenotype characterized by dwarfism, failure to germinate and failure to flower normally. This phenotype can be reverted to wild type by application of GAs (Koornneef et al., 1990; Jacobsen and Olszewski, 1991). Although flower initiation in the mutant occurs normally, anther development in the mutant becomes arrested at a bud length of 2.5 mm, which is before the initiation of meiosis (Jacobsen and Olszewski, 1991). The anthers remain developmentally arrested and remain responsive to GA treatment, until the bud reaches a length of 3.7 mm. The pollen mother cells in these arrested anthers are in the G1 phase of the premeiotic interphase, while outer and inner tapetum cells are at the uninucleate and binucleate stages, respectively (Jacobsen and Olszewski, 1991). Application of GA also causes specific changes in the gene expression pattern in the developmentally arrested anthers (Jacobsen et al., 1994).
To study the molecular mechanisms of GA action during anther development in tomato and to elucidate the cellular basis of the failure of flower development in the mutant, a project to isolate GA-upregulated genes by performing a differential screening of a gib-1 anther cDNA library has been initiated. Besides using the isolated genes as molecular markers for GA response, the gene products can provide an insight into the developmental processes during early flower development in which GAs are involved. This approach can also generate information relating to the role of GA, in combination with ABA, in the regulation of defensins that are known as defence-related proteins. Defensins have been shown to be toxic in vitro to plant pathogenic bacteria and fungi (reviewed in Bohlmann and Apel, 1991; Florack and Stiekema, 1994) and ABA has been shown to induce several wound-inducible genes which are not induced in an ABA-deficient mutant (Hildmann et al., 1992). This has led to the hypothesis that ABA may play a central role in the molecular mechanism by which wounding induces gene expression (Peña-Cortés et al., 1989; Hildmann et al., 1992).
In this paper, the isolation of a cDNA clone corresponding to a GA3-upregulated gene is described, the temporal and tissue-specific expression of the gene and the localization of the gene expression in the mutant and wild type plant. The putative function of the protein is discussed.
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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 with 1x10-3 M GA3 the mutant seeds were germinated in Petri dishes and then transferred to soil. The wild-type tomato (Lycopersicon esculentum 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)).
RNA and DNA extraction
Total RNA was isolated from plant tissues using the method developed previously (Van Eldik et al., 1995). Gibberellic acid was applied directly to developmentally arrested gib-1 flower buds (50 ng GA3 bud-1) or to leaf no. 6 (40 ng GA leaf-1) of 2.5-month-old plants, which contained nine leaves.
To determine the GA3increased accumulation in the wild-type leaf after 6 h or the GA-regulated expression in the mutant leaf by different GAs after 4 h and 8 h, the internodal leaf no. 6 was placed in a 10 µM GA solution. As control, a leaf was placed in water for 6 h and 8 h, respectively.
Regulation of the expression by ABA and GA3, or in combination, was investigated through placing internodal leaf no. 6 of the mutant in the following solution for 8 h: (a) 10 µM GA3; (b) 50 µM GA3; (c) 10 µM GA3 and 50 µM ABA; (d) 10 µM GA3 and 100 µM ABA or (e) 50 µM ABA.
For analysis of the enhancement of the expression by different stress stimuli, leaf no. 6 of 2.5-month-old plants was treated for 8 h as follows: placed in water (a) with or (b) without being pierced by a syringe; (c) 5 min dehydrated under vacuum and left on the bench; (d) placed in water by 4 °C; (e) placed in water under a constant flow of ethylene (5 µl l-1) or (f) 10 µM GA3.
High molecular weight DNA was extracted from young leaves (Bernatzky and Tanksley, 1986).
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 application of 50 ng GA3 bud-1. cDNAs were synthesized from total RNA, using an 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. The cDNA probes were prepared from total RNA of gib-1 anthers 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). Radiolabelled 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 clone was carried out by using the PRISM Sequencing kit (Perking Elmer). Computer analysis of both nucleotide and deduced protein sequence was performed using the University of Wisconsin Computer Group programs (Devereux et al., 1984). The alignment was obtained with Vector NTI.
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).
After prehybridization of the blots in hybridization buffer (6xSSC, 0.5% SDS, 100 µg ml-1 denatured herring sperm DNA, and 5xDenhardt's [1xDenhardt's is 0.02% Ficoll, 0.02% PVP and 0.02% BSA]) for more than 4 h at 55 °C, hybridization was performed for 20 h at 55 °C with radiolabelled probe of tgas118 in hybridization buffer (Van den Heuvel et al., 1999). Washing was carried out at 55 °C with a solution of 1xSSC and 0.1% SDS. The filters were exposed to Kodak X-omat AR or Valva HPX44 X-ray films with an intensifying screen at -80 °C. The radiolabelled probe was removed from the filter by three times washing with a boiling 0.05xSSC and 0.01 M EDTA solution. For confirming the equal amounts of total RNA per lane the gel was monitored under UV light. In addition, where stated, the Northern blot was hybridized with a radiolabelled 28 S ribosomal RNA cDNA fragment isolated from Nicotiana tabacum cv. Petit Havana.
Genomic DNA (40 µg) was digested with EcoRI, HindIII or SacI and electrophoretically fractionated on 1% agarose gels before transfer to Hybond-N as described earlier (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 tgas118 probe in hybridization buffer. Washing was performed at 62 °C with a buffer consisting of 0.5xSSC and 0.1% SDS. The films were exposed to Valva HPX44 or the more sensitive Kodak X-omat AR X-ray films with an intensifying screen at -80 °C.
The autoradiographs were scanned with a Bio-Rad imaging densitometer and the tgas118 signals were quantified with Molecular Analyst software (Bio-Rad, USA). The value for intensity is the density of the band, which is, corrected for the density of the 28 S ribosomal band, relative to the highest signal detected on that blot (which value is set at 100%).
In situ hybridization
The wild-type flower buds at 3 mm length and gib-1 buds 8 h and 24 h after GA3 or H2O treatment 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 cDNA tgas118 cloned in pBluescript II SK(-) were prepared by using (T3/T7) DIG RNA Labelling Kit (Boehringer Mannheim) according to the protocols of the manufacturer. A probe concentration of 40 ng ml-1 was used for overnight hybridization at 42 °C. After washing the detection reaction was performed at room temperature with anti-DIG-AP (Boehringer Mannheim) according to the manufacturer's instructions.
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Results |
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Cloning and genomic organization of tgas118 cDNA
The cDNA clone tgas118 was isolated by differential screening of a gib-1 anther cDNA library, that was constructed 48 h after GA3 treatment of the developmentally arrested buds, to identify clones corresponding to GA3-upregulated mRNAs. By this procedure, a full-length cDNA clone (tgas118) of 469 bp was isolated (EMBL accession number AJ133601) (Fig. 1
). Nucleotide sequence analysis revealed two AATAAA sequences located 50 and 195 bp downstream of the first termination codon which correspond to potential polyadenylation signals according to the consensus described previously (Joshi, 1983). An open reading frame of 216 nucleotides encodes a putative protein of 72 amino acids with a predicted molecular mass of about 8.0 kDa. The amino acid sequence was compared with those of other plant proteins. Alignment of the TGAS118 protein revealed a well-distributed homology with defensins all along the primary structure and conservation of the distribution of the eight cysteine residues in the protein. The TGAS118 protein showed most significant homology with p322 (77% identity), a tuber-specific potato gene (Stiekema et al., 1988), PPT (72% identity), a petunia predominantly pistil-expressed defensin (Karunanandaa et al., 1994), and J1 (69% identity), a fruit specific defensin of bell pepper (Meyer et al., 1996) (Fig. 2). In the barley aleurone layer a GA repressible defensin is reported which shows 32% identity (Heck and Ho, 1996). The TGAS118 protein shares 34% identity with TTP3, a flower-specific defensin of tomato (Milligan and Gasser, 1995) which is larger in size (105 amino acid residues) and contains an acidic C-terminal domain that is absent in TGAS118. Therefore, TGAS118 is structurally different from TPP3.
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Analysis of the encoded protein sequence indicates that it contains a hydrophobic putative signal peptide together with a predicted potential cleavage site located between amino acids 16 and 17 obeying the (-3;-1) rule (Von Heijne, 1986
). All reported defensins contain a signal peptide-like TGAS118, indicating that all of these proteins are secreted. The mature TGAS118 protein, consisting of 56 amino acids, has a calculated molecular mass of 6.2 kDa and is rich in cysteine (14.3%) and arginine (14.3%) residues. Like all other defensins, the mature TGAS118 domain, with a pI of 8.23, is basic and has a net charge of +5.
The presence of tgas118-related genes in tomato has been investigated by Southern analysis. Genomic DNA was isolated from Lycopersicon esculentum cv. Moneymaker and digested with EcoRI, HindIII or SacI, which did not have an internal restriction site in the tgas118 cDNA. As shown in Fig. 1
, a DNA blot probed with the 32P-labelled tgas118 cDNA at moderate stringency revealed in each lane one strong hybridizing band and one or more fragments with weaker hybridization signals, indicating lower homology to the tgas118 probe. These fragments could represent genes that are variants of the tgas118 (Epple et al., 1997). Therefore, it is likely that tgas118 belongs to a small gene family.
Tissue specificity and developmental regulation of tgas118 mRNA expression
Northern blot analysis was performed to determine the tissue-specific expression of the tgas118 gene in the wild-type tomato (Fig. 3). The tgas118 cDNA hybridized specifically to a transcript of approximately 600 nucleotides in length and hybridization was more abundant in the flower than in root and stem. Extending the exposure revealed that a transcript was also present in leaf and fruit (data not shown). In the wild-type flower the tgas118 mRNA was expressed predominantly in the anthers and to a lesser degree in petals and pistil. A weak signal was also found in the sepals. Whether these weaker band are the results of cross hybridization with mRNAs from related genes can not be excluded.
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In addition to the spatial regulation, the tgas118 mRNA expression was developmentally regulated in flower and leaf (Fig. 4A
, B). During flower development the transcript was already detectable in 2 mm wild type and gib-1 buds (Fig. 4B). The accumulation of tgas118 mRNA in wild-type buds increased in 4 mm buds and remained at a high level up to the time when flowers reached 9 mm before declining until anthesis. Contrary to the expression in wild-type buds, the tgas118 transcript diminished in the untreated gib-1 buds during flower development (Fig. 4A). Without GA treatment transcription was detectable in buds up to 6 mm in size, after which the buds aborted. In wild-type leaves tgas118 gene expression was maximum in the youngest tissues (Fig. 4B, lane 9), declining to a basal level of expression in older leaves (Fig. 4B, lane 1). A similar expression pattern was observed during leaf development in gib-1 plants, but the accumulation in the youngest leaf was less abundant than in the wild type.
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GA-regulated expression of tgas118 mRNA
In order to determine the kinetics of the GA3-stimulated tgas118 mRNA accumulation in mutant flower buds and leaf tissues, a time-course experiment was performed (Fig. 5A, B). After application of GA3 to developmentally arrested gib-1 flower buds, tgas118 mRNA abundance was increased 3-fold after 8 h and was enhanced 6-fold 48 h post-treatment (Fig. 5A). In gib-1 leaf a 2-fold enhancement of tgas118 mRNA was observed 8 h after treatment (Fig. 5B). Contrary to the kinetics of accumulation in the flower the GA3increased expression showed to be transient in the leaf and after 24 h the hybridization had declined to a basal level. The stimulatory effect of GA3 treatment on the tgas118 mRNA abundance in gib-1 bud and leaf tissue (Fig. 5, left panels) was also observed in wild-type material after 48 h and 6 h, respectively (Fig. 5, right panels).
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In tomato endogenous GAs are synthesized via the early-non-hydroxylation and early-13-hydroxylation pathway (Koshioka et al., 1994
). The stimulatory effect of different GAs, which are representatives of these biosynthetic pathways, on the abundance of the tgas118 transcripts was studied in the mutant leaf. The gib-1 leaf was placed in 10 µM GA1, GA3, GA4 or GA9 solution. To diminish the possible effect of interconversion of the different GAs, total RNA was isolated 4 h and 8 h after treatment and probed with 32P-labelled tgas118 (Fig. 6). Compared to the control, an increase in the abundance of tgas118 mRNA was found 8 h after treatment with GA1 and GA3 of the early-13-hydroxylated pathway and GA9 of the early-non-hydroxylated pathway, while GA4, which is also synthesized via the early-non-hydroxylated pathway, showed no detectable enhancement.
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Regulation of tgas118 mRNA accumulation by other stimuli
In addition to GA, other growth regulators, like abscisic acid (ABA), might influence tgas118 gene expression. However, it was reported that, although in short-term experiments tomato plants do not respond well to spraying with ABA, a response to this PGR is observed if leaves are fed through the petioles (Peña-Cortés et al., 1989). Therefore the effect of ABA treatment on tgas118 gene expression has been tested by placing the freshly cut gib-1 leaves for 8 h in solutions containing GA3, GA3+ABA or ABA. Figure 7 shows that the accumulation of tgas118 mRNA increased as the GA3 concentration was elevated. Treatment with ABA resulted in a further enhancement of tgas118 mRNA and, depending on the ABA concentration, an additional accumulation was obtained when the mutant leaf was treated with GA3 and ABA together (Fig. 7).
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Also stress factors induce tgas118 expression since enhancement of tgas118 mRNA abundance was also obtained 8 h after dehydration or wounding of the gib-1 leaf, as shown in Fig. 8
. Wounding of the leaf with a syringe resulted in a 3-fold enhancement of the tgas118 mRNA level, while a 2-fold increase was observed after dehydration. Application of ethylene is known to induce gene expression after wounding (reviewed in Kende and Zeevaart, 1997; Morgan and Drew, 1997), however, the gas did not increase tgas118 mRNA accumulation after exposure of the leaf to 5 µl l-1 ethylene.
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Spatial distribution of tgas118 mRNA in flower buds
To localize tgas118 transcripts more precisely in specific tissues and cells within the flower, in situ hybridization was performed with DIG-labelled antisense tgas118 RNA probes (Fig. 9). In 3 mm wild-type flower buds tgas118 mRNA accumulated abundantly in petals, anthers, pistil, and ovary (Fig. 9A, B). In sepals a weak signal of the antisense tgas118 probe was detected, while in petals tgas118 transcripts were detected most prominently in epidermal and parenchyma cells at the edge of the petal. Most abundant expression was found in the anthers, where mRNA accumulation was observed in cells of the tapetum and significant amounts in pollen mother cells, connective tissue and middle layer cells. High levels of tgas118 transcripts were observed in transmitting tissue and cortical cells of the pistil (Fig. 9A) and in ovary in the placenta and the ovules (Fig. 9B).
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In the developmentally arrested gib-1 buds significantly lower levels of tgas118 transcript were observed as compared to the wild type (Fig. 9C
). The tgas118 mRNA abundance decreased further when gib-1 buds lacked any GA treatment (data not shown) which confirms previous results, found after Northern blot analysis (Fig. 4A). Application of GA3 for 8 h resulted in an enhancement of tgas118 mRNA in petals, anthers and pistil (Fig. 9D) which further increased 24 h after treatment (Fig. 9E). The sense tgas118 cDNA probe showed no hybridization in any tissue that was examined (Fig. 9F).
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Discussion |
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As a consequence of GA deficiency in the gib-1 mutant of tomato, the phenotype is characterized by altered leaf morphology and the arrest of flower development. This phenotype can be reverted to wild type by GA application (Jacobsen and Olszewski, 1991
; Koornneef et al., 1990). To study GA regulated gene expression, a GA-regulated cDNA clone, tgas118, was isolated after differential screening of a gib-1 anther cDNA library. With this cDNA clone as probe it was shown that application of GA resulted in an increase of tgas118 gene expression in leaf and flower buds of the mutant and wild type. Based on the amino acid homology, the deduced protein TGAS118 is a defensin, which shows conservation of eight cysteine residues present in the consensus pattern [KR]-x-C-x(3)-[SV]-x(2)-[FYWH]-x-[GF]-x-C-x(5)-C-x(3)-C. Defensins are low-molecular-weight proteins occurring in different tissues of a number of plant species. Recently, a defensin, of which the corresponding gene was repressed by GA, was isolated from the barley aleurone layer. As far as is known, the tgas118 gene represents the first reported defensin up-regulated by GA.
The kinetics of tgas118 mRNA accumulation in gib-1 buds after GA3 treatment is in agreement with the GA3-induced changes in abundance of one class of in vitro translation products in gib-1 anthers previously described earlier (Jacobsen et al., 1994). The kinetics of this class, that exhibited a detectable increase in abundance 8 h after GA3 application and a maximum in abundance at 24 or 48 h post-treatment, were similar to that of tgas118. Normal flower development in the mutant can be induced by a single application of 50 ng GA3 bud-1 (Jacobsen and Olszewski, 1991). This enhances the tgas118 mRNA abundance 6-fold 48 h post-treatment, whereas the localization is similar as in wild-type flowers. In the leaf the GA3 stimulated enhancement of tgas118 mRNA shows a transient expression pattern. Application of GA3 to gib-1 leaves of internode no. 6 increased tgas118 mRNA after 4 h, and the mRNA accumulated 2-fold at 8 h post-treatment, but it declined to levels similar as in untreated leaves 24 h after treatment. The GA3 stimulated tgas118 mRNA accumulation was shown to be concentration dependent in leaf. When higher GA concentrations were used, the enhancement remained temporary (data not shown). Although the reason for this transient expression pattern is unclear, at least one explanation for this result in the leaf can be envisaged. Leaf morphology is affected and the growth rate is reduced during all stages of development in the mutant (Koornneef et al., 1990). Beside a lower abundance in the youngest leaf, the developmentally regulated expression pattern of tgas118 in the mutant leaves is similar as in wild-type leaves. This indicates that the leaf is already differentiated with respect to the tgas118 gene expression. Beside the induction of elongation (Koornneef et al., 1990; personal observations), treatment of the leaf with GA induces no developmental changes which are correlated with an increase of tgas118 mRNA.
Endogenous GAs in tomato fruits are presumed to be synthesized via the early-non-hydroxylation (like GA4 and GA9) and early-13-hydroxylation pathway (like GA1 and GA3) (Koshioka et al., 1994). Of the GAs that have been identified, GA1 is proposed to be the most important for regulating elongation growth (Phinney, 1984), and the early-non-hydroxylated pathway might function in specific organs such as seeds (Kobayashi et al., 1988; Koshioka et al., 1994). In petunia anthers and corollas GA1, GA4 and GA9 are detectable and GA4 is the most abundant. These GAs are effective in stimulating corolla pigmentation and chs gene expression (Weiss et al., 1995). This study's results show that, in the leaf, GA1 is more effective in enhancing tgas118 mRNA abundance than GA3 and GA9, while GA4 lacks the capacity to stimulate the accumulation of tgas118 mRNA.
The extent of the increased accumulation of tgas118 mRNA levels is lower in wild-type flowers than in mutant flowers 48 h post-treatment. An explanation may be that wild-type plants, which contain endogenous GAs, have a reduced sensitivity to applied GA. It might also be possible that GA catabolism is more active in wild-type tissues in response to the endogenous GAs. If this is the case, then less of the exogenously applied GA can act in the wild-type tissue resulting in a reduced response.
Although the biological role for TGAS118 remains to be determined, the structure of the deduced TGAS118 protein suggests that it may behave like a defensin, and act to protect against pathogenic attack (reviewed in Bohlmann and Apel, 1991; Florack and Stiekema, 1994). The fruit-specific defensin of bell pepper which shares 69% identity with the TGAS118 protein, possesses in vitro an antifungal activity (Meyer et al., 1996). The role as defence protein is consistent with the accumulation of tgas118 mRNA, which is developmentally regulated and controlled by environmental signals. A central role in the signal transduction pathway of this response could be ABA and not ethylene. Several wound-inducible genes accumulate after treatment with ABA and are not induced in an ABA-deficient mutant (Peña-Cortés et al., 1989; Hildmann et al., 1992). A model in which ABA and GA regulate the tgas118 expression is attractive, because both ABA and GA levels are affected by the amount of environmental stress experienced by the plant. In soybean seedlings, levels of GA1 decrease with increasing stress while ABA increases under these conditions (Bensen et al., 1990). Thus ABA may control the response to phytopathogen attack, when the endogenous GA levels decreases.
In the flower the tgas118 mRNA is predominantly localised in the anthers, pistil, and ovary. Although the TGAS118 protein appears to have a signal sequence as in other defensins, it is unknown whether TGAS118 is secreted to the cell wall or remains within an intracellular compartment like the vacuole (reviewed in Bohlman and Apel, 1991; Florack and Stiekema, 1994). The high abundance of tgas118 mRNA in young leaves and in reproductive organs of the flower could indicate that it protects these tissues against potential attack by pathogens. In addition, if the accumulation of tgas118 mRNA in older leaves after wounding and dehydration should result in an enhanced protein synthesis, this might result in an enhanced resistance against phytopathogens. Defence-related proteins have been grouped into three classes (Bowles, 1990), namely those that affect the extracellular matrix and therefore have a passive role in defence, those that appear in relation to a defence response, but whose function is unknown, and those that act directly as deterrents. The latter group includes proteins that exhibit antimicrobial activities or catalyse the synthesis of antimicrobial products. Wound-inducible proteinase inhibitors belonging to this class, like defensins, are found in both leaf and flower tissues (Hildmann et al., 1992) showing that a defence mechanism is present in these organs in which TGAS118 may also be involved.
These results show that the mechanism by which GA enhances the tgas118 gene expression in tomato leaf and flower, differs from the mechanism that operates in the aleurone layer of germinating barley where a defensin gene is repressed by GA (Heck and Ho, 1996).
In conclusion, tgas118 is a very useful molecular marker for the elucidation of complex interactions that occur in plants as a response to endogenous and environmental factors, and for studying the role of plant growth regulators in the regulation of gene expression. In addition, the TGAS118 protein may be useful in the study of the role of defensins in plant : microbe interactions.
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Acknowledgments |
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We would like to thank Dr SE Jacobsen and Dr NE Olszewski for providing the gib-1 anther cDNA library. We also thank 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).
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Notes |
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1 The nucleotide sequence data is reported in the EMBL Nucleotide Sequence Database under the accession numbers AJ133601 (tgas118).
2 To whom correspondence should be addressed. Fax +31 24 3652787. E-mail: wullems{at}sci.kun.nl
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