JXB Advance Access originally published online on July 25, 2005
Journal of Experimental Botany 2005 56(419):2409-2420; doi:10.1093/jxb/eri234
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RESEARCH PAPER |
The Arabidopsis mutant eer2 has enhanced ethylene responses in the light
Unit Plant Hormone Signaling and Bio-imaging, Department of Molecular Genetics, Ghent University, KL Ledeganckstraat 35, B-9000 Gent, Belgium
To whom correspondence should be addressed. Fax: +32 9 264 53 33. E-mail: dominique.vanderstraeten{at}ugent.be
Received 12 May 2005; Accepted 25 May 2005
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Abstract |
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By screening for ethylene response mutants in Arabidopsis, a novel mutant, eer2, was isolated which displays enhanced ethylene responses. On a low nutrient medium (LNM) light-grown eer2 seedlings showed a significant hypocotyl elongation in response to low levels of 1-amino-cyclopropane-1-carboxylate (ACC), the precursor of ethylene, compared with the wild type, indicating that eer2 is hypersensitive to ethylene. Treatment with 1-MCP (1-methylcyclopropene), a competitive inhibitor of ethylene signalling, suppressed this hypersensitive response, demonstrating that it is a bona fide ethylene effect. By contrast, roots of eer2 were less sensitive than the wild type to low concentrations of ACC. The ethylene levels in eer2 did not differ from the wild type, indicating that ethylene overproduction is not the primary cause of the eer2 phenotype. In addition to its enhanced ethylene response of hypocotyls, eer2 is also affected in the pattern of senescence and its phenotype depends on the nutritional status of the growth medium. Furthermore, linkage analysis of eer2 suggests that this mutant defines a new locus in ethylene signalling.
Key words: Arabidopsis, enhanced response, ethylene, hypocotyl, metal uptake, metal transport, senescence
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Introduction |
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The gaseous hormone ethylene influences many aspects in the growth and development of plants, including germination, cell elongation, flower and leaf senescence, fruit ripening, sex determination, and defence responses (Yang and Hoffman, 1984
). Many components of ethylene signalling have been identified, mainly through the use of the triple response (Guzman and Ecker, 1990). Indeed, dark-grown seedlings of Arabidopsis undergo conspicuous morphological changes in the presence of ethylene, consisting of a radial swelling of the hypocotyl, an exaggeration in the curvature of the apical hook, and an inhibition of cell elongation in the hypocotyl and root. These morphological alterations have been termed the triple response. Three classes of ethylene response mutants were identified: insensitive mutants, constitutive response mutants, and those that are affected in an organ-specific manner (Bleecker et al., 1988; Kieber et al., 1993; Van Der Straeten et al., 1993; Roman et al., 1995). Cloning and characterization of genes of the ethylene signalling pathway provided insight into the molecular mechanisms responsible for a plant's response to the gaseous hormone. The ethylene signal transduction chain in Arabidopsis, as presently conceived, consists of five partially functionally redundant receptors, which negatively regulate ethylene signalling (Bleecker et al., 1988; Chang et al., 1993; Hua et al., 1995, 1998; Hua and Meyerowitz, 1998; Sakai et al., 1998). These receptors have significant similarity to His protein kinase receptors of two-component regulatory systems (Bleecker, 1999). Ethylene binding requires a copper cofactor as part of the functional receptor (Rodriguez et al., 1999), and a copper transporter protein, RAN1, is thought to provide copper ions to the receptors (Hirayama et al., 1999; Woeste and Kieber, 2000). Downstream of the ethylene receptors is CTR1, a Raf-like mitogen-activated protein kinase kinase kinase (MAPKKK) (Kieber et al., 1993). Direct evidence that a MAPK cascade is part of the ethylene signal transduction pathway in plants has been provided recently (Ouaked et al., 2003). EIN2 acts downstream of CTR1; its amino-terminal half encodes an integral membrane protein that exhibits significant similarity to the Nramp family of cation transporters (Alonso et al., 1999). In response to ethylene, plants modulate the expression of specific genes at the transcriptional and post-transcriptional levels (Lincoln and Fischer, 1988; Zegzouti et al., 1999; Koyama et al., 2001). Responses downstream of EIN2 are modulated by a two-step cascade of transcriptional regulators involving two families of transcription factors, the EIN3 (ethylene insensitive)/EILs (EIN3-like) proteins and the ERFs (ethylene response element binding factor) (Chao et al., 1997). EIN3 is a nuclear-localized DNA-binding protein belonging to a small multigene family in Arabidopsis. The stability of EIN3 plays a central role in ethylene response and is regulated through the F-box proteins EBF1 and EBF2 (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004). Two EIN3-like proteins, EIL1 and EIL2, could complement a loss-of-function mutation in EIN3, indicating that they are also involved in ethylene signal transduction. The immediate target of EIN3 is ERF1 which contains a primary ethylene response element (PERE) in its promotor (Solano et al., 1998). ERF1 belongs to a large family of plant-specific transcription factors referred to as ERFs, which bind to the GCC box and activate the expression of secondary ethylene-response genes.
To date, the standard triple response screen is probably saturated for the identification of viable loss-of-function mutants. However, refinements of the screen continue to yield results. One method consisted in the screening for mutations that display an enhanced ethylene response at a low ethylene concentration. Using this screen, the enhanced-ethylene-response (eer1) mutant was isolated (Larsen and Chang, 2001). Molecular cloning of the eer1 mutation revealed that it is a new allele of RCN1, an A regulatory subunit of PP2A (Larsen and Cancel, 2003). Furthermore, five components of the ethylene-response pathway were identified by screening at a low concentration of the ethylene precursor (wei1-wei5, weak ethylene-insensitive mutants) (Alonso et al., 2003). Alternative screens might help to identify new ethylene-related loci apart from those implicated at the etiolated seedling stage. Two methods are based on the ethylene response in the light of nutrient-deficient seedlings at different stages of development. The first derives from the fact that ethylene induces hypocotyl elongation in the light (Smalle et al., 1997). The second on the observation that ethylene stimulates leaf emergence (Van Der Straeten et al., 1999). Using these novel assays, additional ethylene-related loci were discovered (Van Der Straeten et al., 1999; Vandenbussche et al., 2003).
Here eer2, a novel member of the class of enhanced ethylene response mutants, is described. The mutant displays a measurable ethylene response upon exposure to levels of ethylene that are insufficient to trigger a response in the wild type. Furthermore, eer2 is affected in its senescence pattern and its phenotype depends on the nutritional status of the growth medium. Possible roles for EER2 are discussed.
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Materials and methods |
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Plant materials and growth conditions
Approximately 33 000 fast neutron (FN) mutagenized M2 Columbia seeds (Lehle seeds) were screened. Columbia (Col-0) was purchased from Lehle seeds (Round Rock, TX). The wild-type Ler-0 (Landsberg erecta) as well as the ethylene response mutants etr1-3, ctr1-1, and ein2-1 originated from the Arabidopsis Biological Resource Center (ABRC) at Ohio State University. The eer2 mutant was backcrossed twice to the Col-0 wild type. Conditions of the growth chamber and greenhouse were 22 °C and 60% relative humidity, white fluorescent light (75 µmol m–2 s–1), and long day conditions (16/8 h light/dark).
Media and treatments
The Arabidopsis seedlings were grown under sterile conditions as described in Smalle et al. (1997). The rich medium used was GM (Growth Medium) supplemented with 0.5 g l–1 of MES (Roman et al., 1995). CuSO4 was obtained from Vel (Leuven, Belgium), ZnSO4, MnCl2, and CaCl2 from Sigma-Aldrich (St Louis, MO), and K2SO4 and CuSO4 from Merck (Darmstadt, Germany). ACC (1-aminocyclopropane-1-carboxylic acid) and aminoethoxyvinylglycine (AVG) were obtained from Sigma-Aldrich (St Louis, MO). All hormone and inhibitor solutions were added to the medium after filter sterilization. Plates were stored at 4 °C in the dark for 2 d and then put in a growth chamber. 1-MCP was supplied by the Department of Organic Chemistry (Ghent University, Ghent, Belgium). For 1-MCP gassing, seedlings were grown in 30 µmol m–2 s–1 photosynthetic photon flux density for 8 d. Treatment with 1-MCP (250 ppm) was performed for 20 h d–1. Flushing of the growth chamber occurred during the subjective morning for 4 h at a flux corresponding to four refreshments per hour. For the northern blot analysis, seedlings were grown on GM medium at 60 µmol m–2 s–1 photosynthetic photon flux density. At 15 d, plantlets were treated five times with 250 ppm 1-MCP for 6 h d–1 in a sealed container on a daily basis.
Biometric analysis
Measurements of hypocotyl length of light-grown seedlings were taken as in Smalle et al. (1997) using a Stemi SV 11 microscope equipped with a graduated ocular (Zeiss, Jena, Germany). The rosette diameter was measured after full leaf expansion using a ruler with 1 mm precision. The number of leaves was counted at flowering time (the day the first flower was open). Measurement of the inflorescence, branching, peduncle and silique heights, as well as the number of siliques and the number of seeds per silique were performed on dry plants. Surface area measurements of leaf 5 and leaf 8 after full leaf expansion were obtained by taping the respective seedlings on Whatmann 3MM paper, followed by scanning and subsequent analysis of the images using Scion Image software (Scion Corporation, Frederick, MD).
Ethylene measurements
For ethylene measurements, wild-type and eer2 seeds were sterilized and sown on GM medium. The seedlings were grown for 19 d, 5–10 seedlings were harvested and capped in a 10 ml vial. Emanation of ethylene was measured after 2 d by gas chromatography (flow-through system) as described previously by Pham-Tuan et al. (2000). The measurements were repeated three times, each measurement with six samples per line. All measurements were performed at the same time of the day. Settings: inlet: 150 °C, 11.50 psi, 9 ml min–1; column: 11.50 psi, 6 ml min–1; oven: 180 °C.
Ethylene treatments
Wild type (Col-0), and ethylene response mutants were sown under sterile conditions (Smalle et al., 1997) on GM. At 19 d after sowing, the plants were placed inside a chamber dedicated to gas exposures. Subsequently, 1 ppm of ethylene or air (organic carbon free, Air Liquide Belge NV, Aalter, Belgium) was flushed through for 24 h at a flux rate of 250 ml min–1. Experiments were repeated three times independently. Ambient conditions were 22 °C, 60% humidity, and white fluorescence light (75 µM m–2 s–1) under long-day conditions (16/8 h light/dark).
RNA preparation and gel blot analysis
For the analysis of GST2 expression in wild-type and ethylene mutants, total RNA was extracted using TrizolR reagent (GIBCO/BRL, Gaitherburg, MD) according to the manufacturer's instructions. 30 µg of total RNA was denaturated and loaded on 1.2% (w/v) agarose gel. Gel- and running buffers contained 10 mM MOPS/10 mM TEA. The RNAs were blotted to nylon membranes (Hybond N, Amersham, UK) in 25 mM phosphate buffer as described (Sambrook et al., 1989). Blotting was followed by baking the filter at 80 °C for 2 h. Hybridization was at 65 °C following the method of Church and Gilbert (1984). Probes used in this research were the EcoRI insert of a cDNA clone encoding an ethylene-regulated glutathione-S-transferase (Zhou and Goldsbrough, 1993), an Arabidopsis thaliana 18S ribosomal RNA probe to allow normalization, and the CAB2-probe from Arabidopsis ecotype Columbia made by cloning a CAB2 PCR-fragment in the pGemT-vector (primers used: 5'-ACCGCTGGACTTTCAGCTGAT-3'; 5'-AAATACAAACTGATAAAACTT-3'), all 32P-labelled. Radiolabelling was performed by random priming and polymerisation using the T7-Quick Prime Set. Subsequently, the membranes were washed with 2x SSC, 0.1% SDS followed by 0.2x SSC, 0.1% SDS. Filters were exposed to Kodak films (XAR) for about 24 h. The signals were quantified using the ImageQuant program on a phosphorimager (Molecular Dynamics, 445SI).
From MCP-treated samples, RNA was extracted using the Qiagen RNeasy kit. 20 µg of RNA was loaded on gel and, for hybridization, Denhardt's solution was used. All other steps were identical as described above.
Measurement of chlorophyll levels
About 40 wild-type and mutant plants were grown on soil in the greenhouse. Samples of leaves 5 and 6 were taken, starting from 4 weeks after sowing, with intervals of 5 d. At each time point the length of leaves was measured and a leaf disc of about 0.5 cm2 was perforated from the widest part of the leaf blade. For chlorophyll determination, leaf 5 and 6 discs were first ground in liquid nitrogen. This was followed by incubating the pulverized tissue in 80% acetone (500 ml 0.5 cm–2) for 3 h in the dark. After brief shaking, the samples were centrifuged for 5 min at 5000 rpm. The supernatant was measured in the spectrophotometer (DU®-64 Spectrophotometer, Beckman) at 664 nm and 647 nm. Calculations for the chlorophyll a-, chlorophyll b-, and the total chlorophyll amounts were based on the following formulas:
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In addition, a portable pulse amplitude modulated fluorometer (PAM-2000; H Walz) was used to determine effective quantum yield, 
F/F'm (Y) in a similar experiment (Lootens and Vandecasteele, 2000). The light-emitting diode (LED) was positioned at a distance of 3 mm above the adaxial front surface. After 0.6 s, a LED light source was switched on. The quantum yield, F/F'm (Y), was determined over 26 d with a 4 d interval between individual measurements.
Linkage analysis
The increase in hypocotyl length on ACC, the delay in bolting and flowering, and the pale phenotype are traits that co-segregated in a population of 161 F2 plants of a cross between Lerxeer2 in a ratio 126:35 (wild type:mutant); and in a backcross of eer2 with Col-0 in a ratio 69:18 (wild type:mutant). Mapping of the eer2 locus was performed with simple sequence length polymorphism markers (Bell and Ecker, 1994) and amplified fragment length polymorphism markers (Vos et al., 1995). eer2 was crossed to Ler and the F2 population was scored for mutant and wild-type phenotypes. Per F2 individual, DNA was prepared from a single leaf with a single-step protocol (Thomson and Henry, 1995) or the DNeasy mini kit (Qiagen, Hilden, Germany). AFLP analysis was performed according to Vos et al. (1995). A total of 210 F2 individuals were scored. All primers and adaptors were obtained from Genset (Paris, France). Total genomic DNA was digested by the restriction enzymes SacI/MseI. AFLP fingerprints were generated using different primer combinations (markers of chromosome 1 and 4) with selectivity +2/+2. The DNA fingerprints were scored for the presence or absence of bands.
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Results |
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Isolation and phenotypic characterization of eer2
A novel mutant, designated eer2 (enhanced ethylene response), was isolated by screening a light-grown population of mutant seedlings on Low Nutrient Medium (LNM) in the presence of the ethylene precursor ACC (1-aminocyclopropane-1-carboxylic acid). Previous research indicated that the hypocotyl length of wild-type Col-0 seedlings grown on LNM in the light, reaches up to twice the size of the untreated control in the presence of 50 µM ACC (Smalle et al., 1997
). For eer2, the hypocotyl length was almost doubled in the presence of 1 µM ACC compared with seedlings grown in the absence of ACC, whereas only a limited hypocotyl elongation was seen for the wild type under the same conditions (Fig. 1A). Furthermore, the hypocotyl elongation was significantly more pronounced than that of the wild type on media containing 10 µM and 50 µM ACC. In these conditions, the ethylene-insensitive mutant etr1-3 displayed no elongation. From these data it was concluded that light-grown eer2 is more sensitive to ACC than the wild type. It should be mentioned that the hypocotyl of eer2 was longer than that of the wild type even in the absence of exogenously applied ACC (Fig. 1A). Treatment with 1 µM aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis, did not fully revert the eer2 hypocotyl phenotype (data not shown). This may be due to an incomplete inhibition of ethylene production. Alternatively, this may indicate that the eer2 phenotype is caused by a growth effect unrelated to ethylene or that light-grown eer2 has a weak constitutive ethylene response.
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Further experiments on eer2 demonstrated that the hypersensitive effect is specific for the hypocotyl elongation in light-grown seedlings (Fig. 1B). Inhibition of root elongation by ACC was significantly reduced in eer2, while root length in the absence of ACC was similar to the wild type. Figure 1B shows more than 50% inhibition of root growth in the presence of 0.1 µM ACC for the wild type, whereas a similar response is only seen in the presence of 10 µM ACC for eer2. On higher concentrations of ACC the difference in root length between eer2 and wild type was reduced, indicating that higher ethylene concentrations overcome the partial insensitivity of eer2.
In addition, the length of hypocotyls and roots of dark-grown seedlings were measured upon exposure to ACC. Both parameters are hallmarks of the triple response. The response of hypocotyls and roots grown in the dark on Germination Medium (GM) was similar to the wild type (Fig. 2). To determine if the medium composition could have influenced the result, the test was repeated on LNM (data not shown). Here again, eer2 and wild-type responses to ACC were similar; thus the medium has no impact on the phenotype.
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From these results it was concluded that the hypersensitive response is specific for hypocotyls of light-grown eer2 seedlings.
To confirm the ethylene-dependent nature of the eer2 mutation, treatment with 1-methylcyclopropene (1-MCP), which is a competitive inhibitor of ethylene signalling, was performed. As presented in Fig. 1C, the addition of 250 ppm 1-MCP could block the ACC-induced hypocotyl elongation in the wild type and in eer2. The suppression of the hypersensitive response of eer2 in the presence of ACC and 1-MCP supports the fact that the ACC effect was ethylene-dependent. This assumption was confirmed by ethylene treatment of wild-type and eer2-seedlings (Fig. 1D). In the presence of 1 ppm ethylene, eer2 displayed, on average, 74% of hypocotyl elongation, while only ±27% increase was observed for the wild type.
Measurements of ethylene emanation of eer2 were also performed. Plants were grown for 3 weeks on GM. eer2 and wild-type plants accumulated a similar level of ethylene (Table 1).
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Morphology of eer2
The phenotype of 6-week-old eer2 on soil can be seen in Fig. 2A. The most striking aspect is the pale colour of the interveinal regions of the leaf, resulting in a dark-green venation pattern. eer2 also showed a delay in bolting and flowering time compared with Col-0 wild type plants (Table 2). eer2 was, in general, smaller than the wild type, characterized by smaller rosette leaves and a shorter inflorescence stem (Table 2). Interestingly, eer2 rosette leaves were fully yellow when grown on GM after 3 weeks of growth on GM compared with soil-grown plants (Fig. 3B).
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Epinastic response of eer2
Hypersensitivity to ACC is also reflected by the epinastic response of cotyledons. This is a known ethylene response and is clearly seen in the ctr1-1-mutant (English et al., 1995
). In each panel in Fig. 4, the seedlings on the left represent Col-0; the ones on the right are eer2 seedlings, all grown on LNM in the light. In the presence of 1 µM ACC, epinasty of the cotyledons is already visible in eer2, while in the wild type at least 10 µM ACC is necessary to achieve a similar response (Fig. 4A). A comparable difference in epinastic response was seen in the first leaf pair. Furthermore, this response was alleviated by treatment with 1-MCP (data not shown). In the presence of ethylene, an identical phenotypic response could be observed, indicating that the ACC-effect is a true ethylene-effect (Fig. 4B). For the quantification, exaggerated epinasty was defined as cotyledons that were bent such that they touched the hypocotyl (Fig. 4C). Exaggerated epinasty was more frequently observed in the eer2 mutant compared with the wild type, especially in the presence of ethylene, where about 75% of eer2 and only 30% of wild-type seedlings displayed exaggerated epinasty.
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The eer2-mutation influences ethylene-regulated gene expression
To investigate whether the hypersensitivity to ethylene in eer2 was also reflected at the molecular level, the expression of an ethylene-regulated gene, GST2, gluthatione-S-transferase, was analysed (Zhou and Goldsbrough, 1993
). The GST2 expression in eer2 exceeded the level of wild type-control when treated with purified air, a feature reminiscent of ctr1-1 (Fig. 5A). The steady-state mRNA-level for GST2 increased in wild-type plants upon treatment with ethylene, but was not significantly induced in eer2 after treatment. The response of the ctr1-1 mutant to ethylene is noteworthy. Ethylene responsiveness of three ctr1 mutants was also found in terms of hypocotyl shortening and increased radial thickness (Larsen and Chang, 2001).
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To determine whether the GST2 expression in the mutant was due to increased responsiveness to endogenous ethylene or due to a constitutive response, GST2 expression was examined in the presence of the ethylene signalling inhibitor 1-MCP (Fig. 5B). As seen in Fig. 5A, the level of GST2 messenger in eer2 was significantly induced as compared to the wild type (6-fold). In the presence of 1-MCP, the mRNA-level for GST2 was clearly decreased in the wild type, while in eer2 the decrease was insignificant. From these results, it was concluded that GST2 is constitutively expressed in eer2 at this stage of development.
eer2 does not senesce faster than wild type
Given the chlorotic nature of eer2 on GM and soil (Fig. 3), the constitutive expression of GST2, and the described role of ethylene in senescence, the senescence pattern of eer2 was examined. To that end, the total chlorophyll content in excised leaves was measured. Leaves 5 and 6 (fully expanded) of 4-week-old eer2 plants (starting point), grown on soil, showed a significantly lower chlorophyll content than the corresponding wild-type leaves (Fig. 6A). In addition, the course of the chlorophyll loss was less steep in eer2 compared with wild type, indicating that the senescence pattern is altered in eer2 (Fig. 6A). On day 32 after the start of measurements, chlorophyll was not measurable in the wild type. By contrast, eer2 retained approximately 60% of total chlorophyll content relative to the starting point. This delayed senescence in eer2 could be related to the late flowering phenotype of eer2 plants. Wild-type plants started to flower at day 28 after the start of the measurements (4-week-old plants), while for eer2 this was only the case on day 60 after the start of the measurements.
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In parallel, photosynthetically active chlorophyll was quantified. The method is based on an in vivo measurement of chlorophyll fluorescence, represented by the overall quantum yield of photochemical energy conversion, (F'm – Ft)/F'm (see Materials and methods) (Lootens and Vandecasteele, 2000
). The analysis of chlorophyll fluorescence confirmed the results obtained by spectrophotometric measurement of total intact chlorophyll molecules (Fig. 6B).
Supplementation of Hoagland medium with Zn2+ or Mn2+ reduces leaf chlorophyll contents in eer2
By growing the eer2-mutant on both GM and Hoagland medium, a striking difference was noticed. On GM, eer2 demonstrated severe chlorophyll loss after 3 weeks, resulting in yellow leaves (Fig. 7A). Surprisingly, this did not happen when growing eer2 on Hoagland medium (Fig. 7A). In order to identify the factor causing this difference in phenotype, a comparison of both media was made. Since Hoagland only contains mineral salts, those ingredients that are specific for GM were removed one by one. eer2-plants grown on GM-media without myo-inositol, without vitamins, or without sugar (present in GM medium and not in Hoagland medium) still resulted in yellow plants (data not shown). These results suggested an implication of mineral salts in the conspicuous phenotype of eer2 on GM. Adjusting the concentrations of Mn2+ and Zn2+ in Hoagland to those present in GM, resulted in interesting observations. Three-week-old mutant plants grown on Hoagland-medium supplemented with additional Mn2+ (added as MnCl2) displayed a fully chlorotic phenotype (Fig. 6B). The same happened on Hoagland-medium supplemented with additional Zn2+ (added as ZnSO4) (Fig. 7B) In both cases, but especially in the presence of Zn2+, the plants stayed smaller than the control plants without additional metals. This can possibly be explained by the fact that the Mn2+-concentration was increased 10-fold and the Zn2+-concentration about 200-fold as compared with the control Hoagland medium. The plants could support these high amounts of Mn2+ and Zn2+ over 21 d because they fully recovered in soil thereafter. The addition of CuSO4, K2SO4, and CaCl2 resulted in normal green eer2 plants, indicating that the chlorotic phenotype is due to the presence of the cations Mn2+ and Zn2+ and not the anions 
or Cl– (data not shown). In addition, the effect of addition of Mn2+ or Zn2+ on the hypocotyl elongation response on LNM medium was tested. Seedlings grown on LNM, LNM+Mn2+, an LNM+Zn2+ displayed similar hypocotyl lengths without ACC and in the presence of 1 and 10 µM ACC, indicating that the eer2 ethylene phenotype was not a pleiotropic effect from a defect in Mn2+ or Zn2+ transport (data not shown).
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Due to the paler phenotype of eer2 on GM compared with that on Hoagland, the level of CAB2-mRNA (chlorophyll a/b binding protein) was investigated in eer2 on both growth media. The CAB2-expression was dramatically decreased in 3-week-old eer2 plants (Fig. 7C) on GM, whereas the expression was similar in eer2 on Hoagland compared with the wild type; thus reflecting the phenotypic observations.
Map location of the eer2 locus
Using a cross between eer2 and wild-type Landsberg, SSLP (Simple Sequence Length Polymorphism) and AFLP (Amplified Fragment Length Polymorphism) mapping were performed. EER2 was linked to the top arm of chromosome 4 and to the centre of chromosome 1. For chromosome 4, the eer2 phenotype showed 100% linkage with BA-12L (15.2 cM) and nga8 (26.56 cM) (tested on 148 homozygous mutant plants). For chromosome 1, linkage analysis placed the EER2 locus at the position of markers SM19_96.2 (16 318 151–16 318 224 bp, 60 cM) and SM19_106.4 (17 715 935–17 716 018 bp, 68 cM), here again 100% linkage was observed (tested on 145 homozygous mutant plants). This implies that the FN-mutagenized eer2 mutant is probably the result of a chromosomal rearrangement. In addition, these data strongly support the fact that eer2 is a novel locus in ethylene signalling, rather than an allele of eer1, since EER1/RCN1 corresponds to positions 8 951 207–8 955 088 bp (at about 40 cM) on the upper arm of chromosome 1, approximately 7.5 Mbp above the eer2 locus.
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Discussion |
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In this report, a new recessive Arabidopsis mutant showing a hypersensitive response to ethylene has been described. This hypersensitive response was specific to hypocotyls of light-grown seedlings on LNM. Under these conditions, the hypocotyl length of eer2 on 1 µM ACC was almost doubled in length, whereas the wild type required more than 10 µM ACC to achieve the same level of elongation. By contrast, roots of eer2 were less sensitive than the wild type to low concentrations of ACC. The suppression of hypocotyl elongation and the epinastic cotyledon responses by the ethylene antagonist 1-MCP showed that the eer2 phenotype was ethylene-dependent.
Hypersensitive phenotypes were described previously for the eer1 mutant (Larsen and Chang, 2001), for loss-of-function mutations of ETR1 (Cancel and Larsen, 2002), and for the ebf1 and ebf2 mutants (Guo and Ecker, 2003; Potuschak et al., 2003). Thus, the eer2 mutant adds to the class of hypersensitive mutants, a fourth class of ethylene-response mutants, besides the well-known constitutive, insensitive, and ethylene biosynthesis mutants. The eer1 mutant displayed enhanced ethylene responsiveness in both the hypocotyl of dark-grown seedlings and adult inflorescence stems. Molecular cloning of the EER1 gene revealed that it is a new allele of RCN1, an A regulatory subunit of PP2A (Larsen and Cancel, 2003). eer1 maps on chromosome 1 (between CAPS marker m235 and unusual floral organs (ufo)). Loss-of-function mutations in the F-box genes EBF1 and EBF2 resulted in enhanced responses to ethylene in dark- and light-grown hypocotyls and roots. By contrast to eer1, ebf1, and ebf2, the enhanced ethylene response of eer2 hypocotyls was only seen in the light, which underlines the importance of alternative screening methods. Moreover, eer1 overproduces ethylene, which is not the case for eer2. Since no previously characterized ethylene mutants are found at the defined map positions on chromosomes 1 and 4, eer2 corresponds to a new locus in ethylene signalling.
Ethylene-induced hypocotyl elongation of light-grown seedlings is likely to be transduced by the same pathway as the one that controls inhibition of hypocotyl elongation in the dark (Smalle et al., 1997). The opposite effects suggest the involvement of light-regulated upstream or downstream components. Since the hypersensitivity towards ethylene is not seen in the dark, EER2 could be a component involved in this ethylene-stimulated hypocotyl elongation in the light. In this case, the EER2 product is proposed negatively to regulate the ethylene signal in certain ethylene responses in the light and may have an opposite function in roots.
A role for eer2 in senescence?
At later stages of development eer2 was still characterized by enhanced ethylene sensitivity. Cotyledons and the first leaf pair were epinastic and GST2, a known ethylene-responsive gene, was constitutively expressed in untreated 19-d-old eer2 plants grown on GM. Plant GSTs have well-described roles in the detoxification and tolerance of crops to herbicides (Lamoureux and Rusness, 1993). In addition, plant GSTs can also act as glutathione peroxidases (Bartling et al., 1993; Edwards et al., 2000). A regulation of ethylene-induced GSTs during senescence is described (Itzhaki et al., 1994). Interestingly, the eer2 plants were totally yellow when grown on GM medium. In Arabidopsis, visible yellowing is widely used to stage the progression of senescence and correlates with biochemical and molecular changes that occur during this developmental process (Hensel et al., 1993; Lohman et al., 1994). Therefore the high accumulation of GST2 mRNA in eer2 in the absence of ethylene could be linked with the altered senescence pattern. This could also explain the lower expression level of CAB, a marker for the photosynthetic stage of development. Previously, it was shown that leaf senescence is triggered by an age-related decline in photosynthetic processes (Hensel et al., 1993). Ethylene plays an important role in leaf senescence. Genetic evidence for this is given by the etr1 and ein2 mutants which both have a delayed senescence (Bleecker et al., 1988; Grbic and Bleecker, 1995; Chao et al., 1997). Furthermore, two of the four ore (oresara) mutants, showing a delayed senescence pattern, are alleles of the ein2 mutant (Oh et al., 1997). On the other hand, ethylene is neither necessary nor sufficient for the occurrence of senescence. Senescence eventually occurs in ethylene-insensitive mutants, while constitutive ethylene response and overproduction mutants do not show premature leaf senescence. Therefore, ethylene does not directly regulate the onset of leaf senescence; but rather acts to modulate its timing. Jing et al. (2002) further demonstrated that ethylene promotes senescence within a specific age window. On soil, the chlorophyll content of eer2 was significantly lower than the wild type at 4 weeks of age, but the senescence process was progressing much slower than observed in the wild type, even slower than in etr1-3 and ein2-1. These results suggest that EER2 could be a regulator of the senescence programme such that loss-of-function of EER2 provokes precocious age-related signals suppressing photosynthetic-associated genes at the moment that the leaf is not yet primed for senescence. Therefore, in the phase prior to the onset of leaf senescence, ethylene cannot promote leaf senescence in eer2 (similar to a constitutive ethylene mutant).
The typical phenotype of eer2 and the lower total chlorophyll content of 4-week-old plants grown on soil also suggest that the locus may encode a protein that functions in chloroplast development or activity. In the case of the ore4-1 mutant, a T-DNA insertion mutant in the plastid ribosomal small subunit protein 7 (PRPS17), the extended leaf longevity is explained by the reduced functioning of the chloroplast consistent with reduced growth (Woo et al., 2002). Also eer2 displays lower chlorophyll content after full leaf expansion, reduced leaf size, and extended longevity. Since the chloroplast is the major source of energy input for the plant through photosynthesis, a reduced functioning of the chloroplast causes a deficiency in energy metabolism. This reduced metabolism could be responsible for the extended longevity in the mutant. However, eer2 is not an allele of ore4-1, since the latter maps on the bottom of chromosome 1.
Accumulation or translocation of metals affected in eer2?
An interesting observation was that eer2 mutants grown on Hoagland medium for 3 weeks failed to show the yellowish phenotype which was typical for growth on GM medium. The addition of the divalent cations Zn2+ and Mn2+ to Hoagland could induce the chlorotic phenotype. What can be learnt from these observations? Zinc is an essential catalytic component of over 300 enzymes, including alkaline phosphatase, alcohol dehydrogenase, Cu/Zn superoxide dismutase, and carbonic anhydrase. In addition, zinc also plays a critical structural role in many proteins, for example, transcription factors. On the other hand, manganese is required for a number of essential processes, including oxygen evolution in photosynthesis, detoxification, and CO2 fixation in C4 and CAM plants. Thus, both essential nutrients are required for chloroplast-related processes. Mutation in the MAN1 gene (for manganese accumulator) leads to a chlorotic phenotype, dwarfism, and late flowering (Delhaize, 1996). Man1 seedlings accumulate a range of metals, indicating that the man1 mutation disrupts the regulation of metal-ion uptake or homeostasis in Arabidopsis. Similarly, toxicity symptoms of high concentrations of Zn2+ are also characterized by chlorosis (Macnair et al., 1999). Therefore, it is conceivable that metal uptake or translocation is hyperactive in eer2. Little is known about metal transporters in Arabidopsis. Most of the recent studies on genes involved in metal transport are done by functional complementation of yeast mutants disabled in metal-uptake. For Mn2+ and Zn2+ these studies were performed on the smf1 mutant strain (defective in Mn2+ uptake) and on the zrt1zrt2 Saccharomyces cerevisiae mutant (defective in Zn2+ uptake) (Korshunova et al., 1999). The broad substrate protein IRT1 could complement the smf1 mutant. This IRT1 protein also complements the zrt1zrt2 mutant, linking Mn2+ and Zn2+ nutrition. Interestingly, four close homologues of IRT1 (ZIP1, ZIP2, ZIP3, and ZIP4) in A. thaliana have been implicated in the transport of Zn2+ (Grotz et al., 1998). Besides the common function in complementing the zrt1zrt2 mutant, these Zn2+ transporters show unique sensitivities to other metal ions that may reflect differences in their substrate specificities. The Zip family is structurally distinct from other metal ion transporters such as the Nramp proteins recently implicated in divalent cation transport (Belouchi et al., 1997). In Arabidopsis, functional studies have shown that AtNramp metal transporters modulate Cd and Fe toxicity (Thomine et al., 2000). Interestingly, recent evidence showed that upon Fe starvation, AtNramp3 disruption leads to increased accumulation of Mn2+ and Zn2+ (Thomine et al., 2003). The above-mentioned Smf1 protein from yeast is also a member of the Nramp protein family and functions in the transport of a variety of divalent cations such as Mn2+, Zn2+, and Cu2+. None of the above-mentioned mutants maps in the vicinity of eer2.
Previously, a link between metal ion homeostasis and ethylene signalling has been established. The N-terminal half of EIN2, a central transducer in the ethylene-signalling pathway, has significant homology to the Nramp divalent cation transporters (Alonso et al., 1999). Analogies with the yeast glucose sensors led to the proposal that EIN2 might function as a sensor for an upstream signal, presumably divalent cations. However, no metal ion transporter activity has been detected for EIN2. Additional evidence for the importance of metals in ethylene-signalling came with the discovery of the copper requirement of ethylene perception (Rodriguez et al., 1999). Recently, Armengaud et al. (2004) discovered a novel role for jasmonic acid (JA) in nutrient signalling by analysing the transcriptional responses of Arabidopsis seedlings to changing the external supply of the essential macronutrient potassium. Therefore, further research in uncovering a similar ethylene effect in metal ion signalling would be very interesting. In conclusion, characterization of additional eer2 alleles and cloning of EER2 will help to understand the function of EER2 in ethylene signalling.
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Acknowledgements |
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We thank Peter Lootens and Paul Vandecasteele from the Agricultural Research Centre in Merelbeke for the training and use of the portable fluorometer PAM-2000, Mira Haegman and Piet Vankeirsbilck for technical assistance with the ethylene measurements, Jan Goeman and Johan Vandereycken for supplying 1-MCP, Andrea Miyasaka de Almeida for critical reading, and Tom Gerats, Janny Peeters, and Marnik Vuylsteke for helpful discussions. This work was supported by a PhD fellowship to Annelies De Paepe and research grants to Dominique Van Der Straeten (G.0281.98, WO.004.99, and G.0345.02) from the Fund for Scientific Research (Flanders).
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Footnotes |
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* Present address: Department of Plant Systems Biology, Ghent University, Flanders Interuniversity Institute for Biotechnology (VIB), Technology Park 927, B-9052 Belgium.
Present address: Wetenschappelijk Instituut Volksgezondheid Louis Pasteur, Centrum voor Bioveiligheid en Biotechnologie, Juliette Wytsmanstraat 14, B-1050 Brussels, Belgium.
Present address: Department of Agronomy, Kentucky University, 104A Kentucky Tobacco Research and Development Center 0236, Lexington, KY 40506, USA.
Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; AFLP, amplified fragment length polymorphism; AVG, aminoethoxyvinylglycine; CAB, chlorophyll a/b-binding protein; Col, Columbia; ctr, constitutive triple response; cM, centiMorgan; eer, enhanced ethylene response; ein, ethylene-insensitive; etr, ethylene-resistant; GM, germination medium; GST, glutathione-S-transferase; Ler, Landsberg erecta; LNM, low nutrient medium; MCP, 1-methylcyclopropene; SSLP, simple sequence length polymorphism.
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