JXB Advance Access originally published online on June 4, 2004
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Journal of Experimental Botany, Vol. 55, No. 402, pp. 1491-1497, July 2004
Journal of Experimental Botany, Vol. 55, No. 402, © Society for Experimental Biology 2004; all rights reserved
RESEARCH PAPER |
Constitutive expression of EIL-like transcription factor partially restores ripening in the ethylene-insensitive Nr tomato mutant*
1Genetic Engineering Research Centre, College of Bioengineering, Chongqing University, Chongqing 400030, PR China
2Waltham Centre for Pet Nutrition, Waltham-on-the-Wolds, Leicestershire, LE14 4RT, UK
3BBSRC Research Group in Plant Gene Regulation, Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK
To whom correspondence should be addressed. Fax: +44 (0)115 9516334. E-mail: Donald.Grierson{at}nottingham.ac.uk
Received 12 November 2003; Accepted 8 April 2004
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Abstract |
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Climacteric fruit ripening is regulated by the phytohormone ethylene. ETHYLENE-INSENSITIVE3 (EIN3) is a transcription factor that functions downstream from the ethylene receptors in the Arabidopsis ethylene signal transduction pathway. Three homologues of the Arabidopsis EIN3 gene have been identified in tomato, Lycopersicon esculentum, EIN3-like or LeEIL, LeEIL1, LeEIL2, and LeEIL3. These transcription factors have been proposed to be functionally redundant positive regulators of multiple ethylene responses. In order to test the role of such factors in the ethylene signal transduction pathway during ripening, EIL1 fused to green fluorescent protein (GFP) has been over-expressed in the ethylene-insensitive non-ripening Nr mutant of tomato. Increased levels of LeEIL1 compensated for the normally reduced levels of LeEIL1 in the Nr mutant, and transgenic Nr plants that exhibited high-level constitutive expression of LeEIL1GFP phenotypically resembled wild-type plants, the fruit ripened and the leaves exhibited epinasty, unlike Nr plants. The EIL1GFP fusion protein was located in the cell nuclei of ripe tomato fruit. The mRNA profile of these plants showed that the expression of certain ethylene-dependent ripening genes was up-regulated, including polygalacturonase and TOMLOX B. However, not all ripening genes and ethylene responses, such as seedling triple response, were restored. These results demonstrate that expressing candidate genes in the Nr ethylene-insensitive background is a valuable general approach for testing the role of putative downstream components in the ethylene-signalling pathway.
Key words: Arabidopsis, EIN3, ethylene, signal transduction, tomato
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Ethylene controls many developmental processes including seedling growth, abscission, senescence, wounding, and fruit ripening. Genes for putative ethylene receptors were first isolated from Arabidopsis following the identification of the etr1 mutant, an ethylene-insensitive mutant that fails to show the typical ‘triple response’ to ethylene (Chang et al., 1992
). Subsequently, other related receptors were identified from Arabidopsis (Bleecker, 1999). Sequence analysis indicates that these receptor proteins are similar to bacterial receptor histidine kinases involved in a two component signalling mechanism and are thought to operate via a phosphotransfer relay (Bleecker et al., 1998; Chang and Stadler, 2001). In the climacteric fruit tomato, ethylene is perceived by a family of six receptor proteins (Giovannoni, 2001; Ciardi and Klee, 2001). Studies on ethylene perception have demonstrated that ethylene receptors operate by a ‘receptor inhibition’ mode of action, whereby the receptors actively repress ethylene responses in the absence of the hormone and are inactive when bound to ethylene allowing ethylene responses to occur (Hackett et al., 2000; Ciardi and Klee, 2001; Chang and Stadler, 2001). The tomato Never-ripe (Nr) mutant, which is ethylene-insensitive and bears fruit that are impaired in softening and colour change, is unable to perceive ethylene due to a mutation in the NR ethylene-binding domain (Lanahan et al., 1994). Antisense suppression of the mutant NR gene product restores ripening, indicating that the NR receptor is not necessary for ripening, although the mutant NR ethylene receptor represses ripening, confirming the ‘receptor inhibition’ model of receptor function (Hackett et al., 2000). ETHYLENE-INSENSITIVE3 (EIN3), a nuclear-localized protein that exhibits DNA binding activity, has been identified as a positive regulatory downstream component of the ethylene signal transduction pathway in the model plant Arabidopsis (Chao et al., 1997). EIN3 has been demonstrated to target the ETHYLENE RESPONSE FACTOR1 (ERF1) gene by binding to an element in its promoter (Solano et al., 1998). The ERF1 gene codes for a transcription factor which binds to the GCC-box of ethylene-regulated genes, initiating a transcriptional cascade in ethylene signalling (Solano et al., 1998). ERFs can be split into three classes based on their amino acid sequence, and class I and III are thought to function as activators of transcription and class II as repressors (Fujimoto et al., 2000; Ohta et al., 2001). Three EIN3 homologues have been identified in tomato, LeEIL1–3. Antisense experiments have suggested that they are functionally redundant positive regulators of all ethylene responses, indicating that differential regulation of ethylene responses occurs downstream from LeEILs at the ERF level (Tieman et al., 2001). In order to test the proposed model of ethylene signal transduction further and to identify ERFs involved in ripening, LeEIL1 fused to GFP has been constitutively expressed in the Nr background. It was anticipated that if the proposed signal transduction pathway in Arabidopsis functioned during climacteric fruit ripening then over-expression of LeEIL1 would overcome the negative regulation exerted on the pathway by the mutant NR receptor. The results presented here demonstrate that LeEIL1GFP is located in the nucleus. Over-expression of LeEIL1 allows the negative repression caused by the mutant receptor to be bypassed, by activating the ethylene signal transduction pathway downstream, leading to restoration of ripening colour change and expression of some but not all ethylene-dependent ripening-related genes. |
Materials and methods |
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Plant material
All experiments were performed using near-isogenic lines of Lycopersicon esculentum Mill. cv. Ailsa Craig and mutant Nr plants that have been grown at Sutton Bonington, Leics., UK for over 25 years. Transgenic and control plants were grown in 24 cm diameter pots in M2 compost (Levington Horticulture, Ipswich, Suffolk, UK) under identical glasshouse conditions. Plants were watered daily and fed with high-nitrogen liquid fertilizer at regular intervals. Flowers were tagged at anthesis and fruit development recorded as days post-anthesis (dpa). Mature green (MG) fruit were defined as 35 dpa and were characterized as being green and shiny with no obvious ripening-associated colour change. Breaker (B) fruit were defined as showing the first signs of ripening-associated colour change from green to yellow. Fruit of subsequent ripening stages were defined in days post-breaker so that B+4 fruit were orange/red in colour. All plant samples for the preparation of total RNA were taken at the same time each day, frozen in liquid nitrogen and stored at –70 °C until required.
Construction of transgene and plant transformation
The transgene construct (pLeEIL1-GFP) was designed to constitutively over-express a functional LeEIL1 (Tieman et al., 2001), under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter, which was fused to GFP (Haseloff et al., 1997), which had the ER retention signal removed. Throughout, all basic methods were as described by Sambrook et al. (1989).
To construct pLeEIL1-GFP, a 2 kbp BamHI/XbaI PCR fragment, including the full coding sequence was first ligated in the sense orientation between the CaMV 35S promoter and terminator of BamHI/XbaI-digested pDH-GFP to yield pDHLeEIL-GFP. The sense gene was then excised from pDHLeEIL-GFP by partial digestion with EcoRI and ligated into similarly digested pBIN19 to yield pLeEIL1-GFP (Fig. 1).
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Plasmids containing the ligated fragment in the correct orientation were identified by restriction digest analysis and by sequencing. After transfer to Agrobacterium tumefaciens strain LBA4404 (Bevan, 1984
) by the freeze-thaw method (An, 1987), the construct was used to transform tomato cotyledon explants (Bird et al., 1988). Transgenic plants that rooted on kanamycin were transferred to compost and grown as described above.
Extraction and analysis of RNA
RNA was extracted from tomato fruit pericarp and leaf as previously described (Smith et al., 1988) except that contaminating carbohydrates and DNA were removed by differential precipitation of the RNA from 4 M LiCl at –20 °C for 1 h. RNA was quantified by spectrophotometry and, following formamide denaturation, 20 µg samples and RNA size markers (GIBCO BRL Life Technologies, Inchinnan, Paisley, UK) were fractionated in 1% (w/v) agarose gels containing 3% (v/v) formaldehyde. RNA was capillary-blotted onto GeneScreen Plus (NEN Life Science Products, Hounslow, UK) membranes which were then prehybridized at 65 °C in 5x SSPE (1x SSPE=150 mM NaCl, 10 mM NaH2PO4, and 1 mM Na2EDTA, pH 7.4), 1% (w/v) SDS, 0.1 M phosphate buffer (pH 6.8), 10% dextran sulphate, 50% formamide, 0.01% sodium pyrophosphate and 150 µg ml–1 sheared, denatured, salmon sperm DNA for 4 h. The RNA was hybridized at 42 °C in the same buffer to 32P-labelled probes generated from pGFP, pLeEIL1, pPG, pRIN, pACO1 or pLoxB cDNA sequences using the Rediprime labelling system from Amersham International, Little Chalfont, Buckinghamshire, UK. After hybridization, membranes were washed in 0.2x SSPE, 0.1% (w/v) SDS at 42 °C and were autoradiographed.
Extraction and analysis of genomic DNA
Genomic DNA was extracted by grinding 5 g of young leaf tissue in 25 ml of ice-cold homogenization buffer [25 mM TRIS-HCl pH 7.6, 20% (v/v) glycerol, 2.5% (w/v) Ficoll 400, 0.44 M sucrose, 10 mM ß-mercaptoethanol, and 0.1% (v/v) Triton X-100]. The homogenate was filtered through muslin and the nuclei pelleted by centrifugation (1000 g, 4 °C, 15 min). The nuclei in the pellet were lysed at 70 °C in urea buffer [42% (w/v) urea, 25 mM TRIS-HCl pH 8.0, 0.5 M NaCl, 50 mM EDTA, and 1% (w/v) N-lauryl sarcosine] and the DNA allowed to dissolve. The solution was extracted twice with phenol/chloroform (1:1, v/v) and the DNA precipitated from the aqueous phase by the addition of an equal volume of ethanol. The DNA was washed successively with 50 mM potassium acetate in 70% (v/v) ethanol, 70% (v/v) ethanol, and 95% (v/v) ethanol, allowed partially to air dry and was dissolved in sterile distilled water (SDW) containing 10 µg ml–1 DNase-free calf pancreatic RNase A (Boehringer Mannheim UK, Lewes, East Sussex, UK) and stored at 4 °C until required. Individual genomic DNA (30 µg) samples were completely digested with EcoRI, separated in 0.8% (w/v) agarose gels and capillary blotted to GeneScreen Plus (NEN Life Science Products, Hounslow, UK) membranes. Membranes were prehybridized as for northern analysis and the DNA hybridized to probes generated from either the cDNA sequence of pLeEIL1 or from the DNA sequence of the neomycin phosphotransferase gene (nptII) located within the T-DNA borders of pBIN19. Membranes were washed at 42 °C in 0.2x SSPE, 0.1% (w/v) SDS and were autoradiographed.
Confocal microscopy
Two-photon images were taken on a commercial Leica SP2 multiphoton scanning laser microscope. The Leica SP2 is equipped with three visible lasers (Ar, Kr, and He/Ne) as well as a Spectra-Physics Tsunami infrared laser for multi-photon imaging. The Tsunami is pumped by a 15 W millennia Vs pump laser, and may be tuned between 750 and 1050 nm. The tomato fruit were cut into thin slices, by hand, and were then placed on a slide and mounted with a drop of VectashieldTM (Vector Laboratories, Peterborough, UK) containing diamidinophenylindole (DAPI) that stains nuclear material. The tomato slices were then viewed under oil emersion (x100) using multi-photon imaging with the DAPI excited at 367 nm and GFP at 590 nm.
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Results |
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Transformation of Nr mutants
A full-length cDNA clone for LeEIL1 (Tieman et al., 2001
) was isolated by PCR and its identity confirmed by DNA sequencing. The LeEIL1 cDNA was inserted in the sense orientation, downstream of the CaMV 35S promoter and upstream from the GFP coding sequence (Haseloff et al., 1997) without the ER retention signal to produce an in-frame LeEIL1GFP fusion. This DNA cassette (Fig. 1) was then inserted into pBin 19 (Bevan, 1984) and the LeEIL1GFP transgene was introduced into 3-week-old Nr cotyledons by Agrobacterium tumefaciens-mediated transformation. Eight primary transformants were initially regenerated on selective media containing 100 mg l–1 kanamycin and grown to maturity. Levels of LeEIL1GFP in young leaves of each transformed line were analysed by northern blot using LeEIL1 as a probe. Four lines of the primary transformants were identified which exhibited expression of LeEIL1GFP (1551, 1567, 1558, and 1568), with two lines (1551 and 1567) showing high expression in leaves (Fig. 2A). Line 1472 appeared untransformed and lines 1646, 1651, and 1656 showed reduced levels of endogenous LeEIL1 possibly due to sense gene silencing. The endogenous LeEIL1 mRNA was detected as a lower band in Fig. 2 and was generally at a lower level than LeEIL1GFP.
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Analysis of ripening in LeEIL1GFP expressing plants
Nr plants are insensitive to ethylene, and fruit do not ripen normally as the NR receptor harbours a mutation in the ethylene-binding domain of the protein. The fruit produced by plants 1551 and 1567 which expressed high levels of LeEIL1GFP fusion mRNA did ripen, although more slowly than wild-type fruit (Fig. 3A). Total RNA was extracted from fruit at the onset of ripening, known as breaker (B) and 4 d after breaker (B+4) and the expression levels of LeEIL1GFP assessed by northern blot. LeEIL1GFP mRNA was expressed at high levels in breaker fruit, but at slightly lower levels in B+4 fruit (Fig. 2B). When ripe, the transgenic LeEIL1GFP fruit visually resembled wild-type fruit and Nr fruit in which the mutant NR receptor had been down-regulated by antisense inhibition (Hackett et al., 2000
). This suggests that the LeEIL1GFP protein is functional and that over-expression compensates for the lack of ethylene signalling exhibited in Nr plants, leading to downstream activation of ethylene responses and overcoming the negative regulation caused by the mutant NR ethylene receptor on the ethylene signal transduction pathway. Visual examination of the transgenic lines expressing LeEIL1GFP indicated that the plants showed typical symptoms of increased response to ethylene: The untransformed Nr control plants showed little or no leaf epinasty, whereas the transgenic Nr plants over-expressing LeEIL1GFP showed enhanced leaf epinasty, indicating that they were responding as if they were sensing ethylene (Fig. 3B). Analysis of the triple response phenotype of seedlings of the To generation of lines 1551, 1567, and 1568, however, showed that ethylene perception and sensitivity had not been restored in seedlings. The transformed lines and the Nr control seedlings showed no triple response when grown with 10 µM ACC (data not shown), unlike Arabidopsis when expression of EIN3 and TEIL (tobacco EIN3-like) gave a constitutive ethylene phenotype (Chao et al., 1997; Kosugi and Ohashi, 2000). This suggests that the presence of LeEIL1GFP was not sufficient, or it was not present at sufficient levels to overcome the negative regulation exerted on the pathway due to the mutant NR receptor. This is consistent with other results from Arabidopsis where constitutive EIN3 expression did not elevate the ERF1 transcription factor to levels observed in the ctr1 mutant (Solano et al., 1998).
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Expression of ripening-related genes
To understand which genes were being up-regulated during the ripening response caused by the expression of LeEIL1GFP, the concentrations of selected ripening-related and ethylene-related mRNAs involved in ripening were examined, including polygalacturonase (PG) (Tucker and Grierson, 1982
; Dellapenna et al., 1986; Montgomery et al., 1993), TOMLOXB (Griffiths et al., 1999), LEACO1 (Holdsworth et al., 1987), and E4 (Lincoln et al., 1987), mRNA levels were analysed by northern blot at breaker and B+4 fruit stages (Fig. 4). PG and TOMLOXB showed higher levels of mRNA in the fruit of plants expressing LeEIL1GFP than in the control Nr plants, although expression levels were not as high as observed in wild type (Ac++) fruit. The PG promoter contains ethylene-inducible elements that have similarity to promoter sequences in the ethylene–dependent genes E4 and E8 (Nicholass et al., 1995) and induction of PG mRNA has been shown to occur at very low levels of ethylene (Sitrit and Bennett, 1998). However, E4 and LEACO1, genes that are known to be up-regulated by ethylene during fruit ripening, exhibited the same expression pattern in transgenic Nr as in control ethylene-insensitive Nr fruit. These results may indicate that E4 and LEACO1 are regulated by a different set of transcription factors compared with PG or that E4 and LEACO1 transcription requires higher levels of LeEIL1 protein. The demonstration that TEIL does not interact with the E4 element (Kosugi and Ohashi, 2000), although the EIN3 binding site shares similarity with a promoter element required for ethylene responsiveness in the tomato E4 gene (Solano et al., 1998), supports the former possibility.
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Location of LeEIL1
LeEIL1 contains the putative nuclear localization sequence, K-R-L-K, which is similar to the sequence identified in Arabidopsis EIN3 (Chao et al., 1997
). This sequence conforms to the minimal nuclear localization sequence identified in other transcriptional regulators (Chelsky et al., 1989). To determine if LeEIL1 is targeted to the nucleus, LeEIL1 was C-terminally tagged with GFP, and the location of the LeEIL3GFP fusion protein was determined by confocal microscopy of transformed Nr B+4 fruit (Fig. 5). The position of the nucleus was confirmed by staining the nuclear material with 4–6-diamidino-2-phenyl indole-2 HCl (DAPI). GFP fluorescence was identified mainly in the nucleus of the Nr plants expressing the LeEIL1GFP fusion protein, although a small amount was detected in the cell cytoplasm. By contrast, plants expressing the GFP protein alone exhibited low levels of fluorescence throughout the cell cytoplasm and no accumulation in the nucleus (Fig. 5). These data indicate that the site of action of the LeEIL1 protein is in the nucleus, which is consistent with what is observed for EIN3 in Arabidopsis and soybean (Chao et al., 1997).
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Activation of other transcription factors
Analysis of the non-ripening tomato mutant rin has shown that LeMADS-RIN is a developmentally-regulated transcription factor that controls fruit ripening. The demonstration that LeMADS-RIN complements the rin mutant has placed LeMADS-RIN upstream of ethylene in the ripening signal transduction pathway and the exogenous application of ethylene does not enhance the expression of LeMADS-RIN (Vrebalov et al., 2002
). However, in the present experiments, LeMADS-RIN expression in LeEIL1GFP Nr transgenic fruit was higher than in Nr control fruit, but somewhat lower than in wild-type control fruit (Fig. 6), suggesting ethylene-related transcription factors may increase RIN mRNA concentration.
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The N-terminal of Arabidopsis EIN3 has been shown to bind to the promoter of ERF1 at –1213 to –1179, and over-expression of EIN3 causes constitutive high-level expression of ERF1 (Solano et al., 1998
). Tomato EILs are thought to be functionally redundant as antisense suppression appears to function in a dose-dependent manner (Tieman et al., 2001). This is similar to results with tobacco where all five NtEILs (Nicotiana tabacum EIN3 like) show no differences in expression levels or tissue specificity during ethylene responses (Rieu et al., 2003). These observations support the suggestion that additional levels of ethylene regulation may occur downstream from EIN3 and that groups of ERF1-like transcription factors may be specific to certain developmental processes. In order to confirm if EILs are functionally redundant or whether there is a defined target for LeEIL1, the class of the ERF1-like transcription factors that are activated by LeEIL1 during ripening were examined in the LeEIL1GFP transgenic plants. The expression levels of Pti4, a class I ERF known to be induced by ethylene and salicylic acid and to play a role in the defence pathway (Zhou et al., 1997; Gu et al., 2002), and a tomato EST with close homology to EFR1 from Arabidopsis, were analysed. The mRNA expression levels of the EST with close homology to ERF1 were increased in the transgenic plants, indicating that EIL1 acts as a transcriptional activator of this gene. However, the expression levels of Pti4 remained unchanged in the ripening LeEIL1GFP transgenic fruit, indicating that LeEIL1GFP was not able to up-regulate Pti4 under the conditions tested (Fig. 6). |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In order to test the hypothesis that LeEIL1, an EIN3 homologue, plays a role in fruit ripening, a LeEIL1GFP fusion has been constitutively expressed in the non-ripening Nr tomato mutant. Expression of LeEIL1GFP was able to restore aspects of ripening in two independently transformed plant lines indicating a role for LeEIL1 in fruit ripening. Analysis of ripening tomato fruit has shown that LeEIL1GFP is located in the nucleus of each cell, as found for the Arabidopsis EIN3 (Chao et al., 1997
). LeEIL1 is 59% identical at the nucleotide level to Arabidopsis EIN3 and the protein is 70% similar. Although they share many structural domains the predicted tomato proteins do not contain the C-terminal asparagine-rich region found in the Arabidopsis genes, which may indicate subtle functional differences (Tieman et al., 2001).
There are three LeEILs in tomato. They have been proposed to be functionally redundant and to regulate ethylene responses throughout plant development, as it is necessary to suppress the expression of all three EILs in order to reduce ethylene sensitivity (Tieman et al., 2001). However, expression of LeEIL1GFP in the Nr background did not restore all ethylene responses, such as the seedling triple response, or up-regulation of all ripening-related genes. This indicates that either LeEIL1GFP was not expressed at a sufficiently high level to restore these functions, that LeEIL2 or LeEIL3 are also required, or that other unknown factors are also affected by the Nr mutation. It is possible that LeEIL1 expression was not restored to levels normally found in wild-type plants, but this seems unlikely, in view of the relative levels of endogenous LeEIL1 and LeEIL1GFP mRNA shown in Fig. 2. Thus, unless the function of LeEIL1 is differentially impaired by the fusion with GFP, these results indicate that more than one factor controls aspects of the ethylene response pathway.
In Arabidopsis EIN3 and EIL1 and EIL2 bind in a sequence-specific manner to the primary ethylene-response element of ERF1, an ethylene-inducible transcription factor, that, in turn, directly binds to the GCC-box of a wide variety of ethylene-responsive pathogenesis-related genes (Solano et al., 1998). The partial restoration of ripening-related genes in these experiments could indicate that only certain ERF transcription factors are dependent on LeEIL1 transcription factor action or that the expression of certain different genes is regulated in a dose-dependent manner. This is the first evidence that an ERF1 like transcription factor is up-regulated due to EIL1 expression (Fig. 6), although an EIN3 to ERF1 to PR proteins pathway has been established in Arabidopsis (Solano et al., 1998; Gu et al., 2000). The up-regulation of RIN (Fig. 6), a MADS box gene known to be the basis of the non-ripening tomato mutant rin, is interesting as this gene has been suggested to function upstream of EIL-like proteins and may indicate a positive feedback loop, as seen in the case of autocatalytic ethylene production. LEACO1, PG, and E4 promoters all contain similar motifs and ethylene-inducible elements (Lincoln et al., 1987; Dellapenna et al., 1989; Nicholass et al., 1995; Montgomery et al., 1993; Blume and Grierson, 1997). However, expression levels of LEACO1 and E4 did not increase in the transgenic LeEIL1GFP plants, despite the fact that the EIN3 binding site in the ERF1 promoter is very similar to that identified in the LEACO1 (Blume and Grierson, 1997) and E4 promoter (Montgomery et al., 1993), whereas PG expression was almost restored to wild-type levels. PG mRNA expression has been shown to occur at a very low ethylene threshold (Sitrit and Bennett, 1998), and the demonstration that PG is up-regulated in the LeEIL1GFP fruit indicates that a direct ethylene pathway from gene expression to ripening, i.e. LeEIL1 to ERF1 to ripening-related proteins is beginning to be elucidated. The fact that E4 and LEACO1 expression is not restored to normal wild-type levels suggests that they are not up-regulated by transcription factors activated by LeEIL1, or that they are required at much higher levels, or that some ripening-related genes respond to or require other transcription factors.
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Acknowledgements |
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This study was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) grant number 42/P09465. We thank Dr SG Kim for help with the fluorescence microscopy.
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Footnotes |
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* Accession numbers: The EMBL data library accession number for the EIL-like transcription factor cDNA nucleotide sequence is AF328784. The TIGR expressed sequence tag (EST) identifier for the EFR1 like transcription factor is EST436804.
Abbreviations: B, breaker; CaMV, cauliflower mosaic virus; dpa, day post-anthesis; ER, endoplasmic reticulum; MG, mature green; Nr, Never-ripe.
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