RESEARCH PAPER |
Ethylene-induced Arabidopsis hypocotyl elongation is dependent on but not mediated by gibberellins
1Unit of Plant Hormone Signaling & Bio-imaging, Department of Molecular Genetics, Ghent University, Ledeganckstraat 35, B-9000 Ghent, Belgium
2Rothamsted Research, West Common, Harpenden, Herts AL5 2JQ, UK
3Université Paris VI, UMR-CNRS 7632, Casier 156, 4, Place Jussieu, 75252 Paris Cedex 05, France
4Penn State University, 25 Yearsley Mill Road, Media, PA 19063, USA
5Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden
* To whom correspondence should be sent. E-mail: Dominique.VanDerStraeten{at}ugent.be
Received 2 August 2007; Revised 20 October 2007 Accepted 22 October 2007
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Abstract |
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Ethylene, or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), can stimulate hypocotyl elongation in the light. It is questioned whether gibberellins (GAs) play a role in this response. Tests with light of different wavelengths demonstrated that the ethylene response depends on blue light and functional cryptochrome signalling. Levels of bio-active GA4 were reduced in seedlings showing an ethylene response. Furthermore, ACC treatment of seedlings caused accumulation of the DELLA protein RGA, a repressor of growth. Concurrently, transcript levels of several GA biosynthesis genes were up-regulated and GA inactivation genes down-regulated by ACC. Hypocotyl elongation in response to ACC was strongly reduced in seedlings with a diminished GA signal, while being vigorously stimulated in a quadruple DELLA knock-out mutant with constitutive GA signalling. These data show that ethylene-driven hypocotyl elongation is mainly blue light-dependent and that this ethylene response, although GA dependent, hence needing a basal GA level, is not mediated by GA, but rather acts via a separate pathway.
Key words: Arabidopsis, ethylene, gibberellin, hypocotyl elongation, light signalling
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDISCUSSIONSupplementary dataReferences |
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Elongation of stem-like organs is influenced by light of various wavelengths and by most plant hormones. Apart from auxins and brassinosteroids, ethylene and gibberellins (GAs) regulate extension growth. The nature of the ethylene response is species-, time-, position-, and condition-dependent (Smalle and Van Der Straeten, 1997; Pierik et al., 2006). For instance, on a low nutrient medium at 21 °C, ethylene stimulates hypocotyl elongation in white light (Smalle and Van Der Straeten, 1997), whereas at 26 °C on richer media, ethylene inhibits growth (Collett et al., 2000). GAs are known principally as growth stimulators (Sponsel and Hedden, 2004; Davidson et al., 2005; Thomas et al., 2005). Their function is reflected in the phenotype of mutants with reduced or enhanced GA biosynthesis or signalling: GA biosynthesis mutants have a dwarfed stature, including short hypocotyls, stems, and petioles. In Arabidopsis, these mutants contain lesions in the ga1 to ga5 genes (Koornneef and van der Veen, 1980; Talon et al., 1990), which encode enzymes catalysing steps throughout the biosynthetic pathway. For example, the GA1 gene encodes ent-copalyl diphosphate synthase (CPS), which catalyses the first committed step of the pathway (Sun et al., 1992; Sun and Kamiya, 1994), while GA5 and GA4 encode a GA 20-oxidase (GA20ox1) and a GA 3-hydroxylase (GA3ox1), respectively, which catalyse the final reactions of the pathway.
Gibberellin signalling in Arabidopsis depends on the DELLA proteins, of which there are five paralogues, GAI, RGA, RGL1, RGL2, and RGL3. While RGA and GAI are most significant for vegetative development (Achard et al., 2003), with RGA having the larger role in elongation growth (Dill and Sun, 2001), RGA, RGL1, and RGL2 function in floral development (Cheng et al., 2004; Tyler et al., 2004). RGL2 also plays a major role in seed germination (Wen and Chang, 2002; Tyler et al., 2004). The function of RGL3 remains unclear, but it is thought to act in seed germination and flower development (Sun and Gubler, 2004; Tyler et al., 2004). Gibberellins destabilize the DELLA proteins, which act as growth repressors, by targeting them for ubiquitination and degradation (Dill et al., 2004). Recently, the identification of soluble GA receptors, including three from Arabidopsis, has indicated that GAs may participate fairly directly in this process since the receptors were shown to interact with the DELLA proteins in a GA-dependent manner (Ueguchi-Tanaka et al., 2005; Griffiths et al., 2006; Nakajima et al., 2006). Events down-stream of the DELLA proteins are less well understood, although several GA-regulated genes have been identified in Arabidopsis, with GASA1 being one of the best characterized (Raventos et al., 2000).
Photomorphogenesis in plants relies at least partly on negative control of GA biosynthesis and/or signalling (Reid et al., 2002; Alabadí et al., 2004; Achard et al., 2007a). Control of hypocotyl elongation by phytochrome has been shown to require a functional GA signalling system (Peng and Harberd, 1997; Achard et al., 2007a); conversely, it was shown that the long hypocotyl phenotype of seedlings deficient in cryptochrome 1 can, at least in the short term, be phenocopied by treatment with auxin and GA (Folta et al., 2003), suggesting that these hormones are important in counteracting the action of blue light. However, little is known about the long-term effects of blue light on GA signalling pathways.
Auxins and GAs are not the only hormones known to stimulate hypocotyl growth in the light. Although ethylene is predominantly known to inhibit elongation, under some conditions it can stimulate elongation growth. In semi-aquatic species, such as Rumex palustris and Ranunculus, ethylene promotes stem growth, as part of a mechanism for flooding tolerance. Deep-water rice varieties have a similar response, in which it was shown that GAs act as mediators between ethylene and elongation growth (Vriezen et al., 2003b). An ethylene-induced elongation response was also observed in certain terrestrial plants (Vandenbussche et al., 2003a, 2005; Pierik et al., 2004). To study this process, the model plant Arabidopsis, in which ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) stimulates hypocotyl elongation in the light, was used (Smalle et al., 1997; De Paepe et al., 2005). This response consists of an increase in hypocotyl elongation at days 3 and 4 after germination, and is absent in gain-of-function etr1 ethylene receptor mutants and constitutively present in mutants of the signalling component CTR1 (Smalle et al., 1997; Saibo et al., 2003). Here it is shown that the stimulation of hypocotyl elongation in response to ethylene in the light is dependent on light quality and fluence rate. It is demonstrated further that blue light perceived by cryptochromes is necessary for this response to occur and that ethylene does not act through additional stimulation of the GA signal in this response.
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Materials and methods |
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Plant materials and growth conditions
Seeds were sterilized in 5% bleach for 15 min and rinsed with sterile water. They were put on low-nutrient agar medium (LNM; Smalle et al., 1997) and left overnight at 4 °C. Seeds were then exposed to white light for 6–18 h to stimulate germination and transferred to the appropriate light conditions. Wild-type Col-0 and Ler-0 seeds, and the mutants ctr1-1, ein2-1, etr1-3, phyA-211, all four in the Col-0 background, and ga1-3, ga5-1, phyB-1 in the Ler-0 background, were obtained from the ABRC; ga1-1 (Ler-0) was obtained from NASC. The pGA1::GUS line (Ler-0) and 3GLG line (Col-0) were gifts from TP Sun and J Mundy, respectively. cry1 and cry1cry2 seeds in the Ler-0 background were as described (Ahmad et al., 2002). Phot1phot2 was in the Col-0 background. etr1-1 seeds were a gift from A Bleecker. 35S::GAI-GFP (Ler-0) and pRGA::GFP-RGA (Ler-0) were gifts from N Harberd and TP Sun, respectively. The ga2ox1-1 mutant in the Col-0 background was obtained from the Arabidopsis Knockout Facility of the University of Wisconsin (WiscDsLox333C08). The ga2ox7-2 mutant in the Col-0 background was obtained from the SALK Institute (SALK_055721; Alonso et al., 2003). Presence of the T-DNA was confirmed by PCR using gene-specific and T-DNA primers (Table 1). Sequencing revealed that in ga2ox1-1 the T-DNA was inserted in exon 2 of GA2ox1, 726 bp after the start codon, and in ga2ox7-2 in exon 1 of GA2ox7, 7 bp after the start codon. Because the insertions are located before (ga2ox7-2) or inside (ga2ox1-1) the part of the genes encoding the catalytic domain, both mutants are expected to be functional-null alleles.
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Paclobutrazol was from Riedel de Haën (Hanover, Germany), ACC from ICN (Aurora, OH, USA), 2,5-norbornadiene from Aldrich (Steinheim, Germany), GA3 and GA4 from Sigma (St Louis, MO, USA).
Light sources
Blue light (470 nm) was produced by Dragon Tape LEDs (Osram, Capelle a/d Ijssel, The Netherlands). Red light was obtained by filtering cool white light (TL, Philips, The Netherlands) through a combination of red plastic filters and red filterfoil. This yielded a spectrum, as measured by an LI-1800 portable spectroradiometer (Li-Cor, Lincoln, NB, USA), with a peak at 640 nm and a lower and higher cut-off at 585 nm and 700 nm, respectively. All treatments of plants were done using continuous lighting.
GA measurements
Arabidopsis seedlings were grown for 3 d in 30 µmol m–2 s–1 of blue light on LNM with or without 50 µM ACC. Two or three (for mutants) and six (for wild type) independent biological replicas were used. They were harvested, frozen in liquid nitrogen, and kept at –80 °C until extraction and analysis. Samples corresponding to 0.5–1 g fresh weight were first homogenized in liquid nitrogen with a mortal and pestle, and thereafter extracted in 3 ml of 80% methanol with 0.02% diethyl dithiocarbamate as antioxidant, using an MM 301 vibration mill (Retsch GmbH & Co. KG, Haan, Germany) at a frequency of 30 Hz s–1 for 3 min after adding 3 mm tungsten carbide beads (Retsch GmbH & Co. KG, Haan, Germany) to each tube to increase the extraction efficiency. Deuterated GAs ([17,17-2H2]GA) were added as internal standards (L Mander, Canberra, Australia) prior to extraction. After centrifugation in an Eppendorf centrifuge for 10 min at 20 800 g, the supernatant was transferred into Kimble tubes and dried under reduced pressure. The residue was redissolved in 50 µl of methanol and 450 µl of H2O (1% HOAc), and the mixture was applied to a C8 (500 mg) ISOLUTE cartridge (Sorbent AB, V. Frölunda, Sweden) previously conditioned with MeOH and equilibrated with 1% HOAc. The cartridge was washed with 10% HOAc, and GAs were eluted with 90% MeOH (1% HOAc). The methanol eluate was dried and, after methylation with ethereal diazomethane, the samples were subjected to reversed-phase HPLC.
The HPLC system consisted of a Waters model 600 pump (Waters Associates, Milford, MA, USA) connected via a Waters 717-autosampler to a 4 µm Symmetri C18 column, 150 mmx3.9 mm i.d. (Waters Associates). The mobile phase was a 20 min linear gradient of 30–100% methanol in 1% aqueous acetic acid at a flow rate of 1 ml min–1. Five fractions corresponding to the GAs of interest were dried, and after evaporation trimethylsilylated in 20 µl of dry pyridine/BSTFA/trimethylchlorosilane (50:50:1, by vol.) at 70 °C for 30 min. The derivatization mixture was then reduced to dryness and dissolved in 15 µl of heptane. Samples were injected in the split-less mode into an HP 5890 gas chromatograph (Hewlett Packard, Palo Alto, CA, USA) fitted with a fused silica glass capillary column (30 m long, 0.25 mm i.d.) with a chemically bonded 0.25 µm DB-5MS stationary phase (J&W Scientific, Folsom, CA, USA). The injector temperature was 270 °C. The column temperature program varied depending on the GA being analysed. The column effluent was introduced into the ion source of a JMS–SX/SX102A mass spectrometer (JEOL, Tokyo, Japan). The interface temperature was 270 °C and the ion source temperature 250 °C. The acceleration voltage was 10 kV, and ions were generated with 70 eV at an emission current of 500 µA. For quantification, samples were analysed in selected reaction monitoring mode (SRM) (Moritz and Olsen 1995). All data were processed by a JEOL MS–MP7010 data system.
Hypocotyl measurements
After the indicated time of growth, seedlings were laid horizontally on an agar plate and hypocotyls were measured using a standard 1 mm scaled ruler. For small seedlings, digital pictures were taken and analysed with NIH ImageJ software (Saibo et al., 2003).
RT-PCR analysis
Seedlings were harvested after 3 d of growth on LNM (Smalle et al., 1997) in 12 µmol m–2 s–1 of blue light, and frozen in liquid nitrogen. Tissue was ground in a ball mill (model MM301; Retsch, Haan, Germany). RNA was extracted using a Qiagen Rneasy mini-kit (Qiagen GmbH, Germany) and samples were treated with DNase (Invitrogen). Reverse transcription (RT) was carried out using the Superscript II system (Invitrogen).
For the semi-quantitative analysis, cDNA was diluted 10 times and 4 µl was used in 20 µl PCR. As controls, non-DNase-treated and non-RT-treated samples were included to confirm the purity of the cDNA. PCRs were performed using specific primers (Table 2). PCR products were visualized on a 1% agarose gel with ethidium bromide staining.
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For quantitative real-time PCR analysis, total RNA (1–2 µg) was treated with DNase I (Turbo DNA-free kit; Ambion, Austin, TX, USA) and 0.5 µg was used as a template to synthesize cDNA using the SuperScript III Platinum Two-Step qRT-PCR Kit with SYBR Green (Invitrogen, Paisley, UK). Gene-specific primers were designed using Primer Express v.2.0 (Applied Biosystems, Foster City, CA, USA) or GENOPLANTE SPADS software (http://urgi.infobiogen.fr/tools/spads/) and are listed in Table 3. PCRs were performed on an ABI 7500 real-time PCR system (Applied Biosystems) using Platinum SYBR Green qPCR SuperMix-UDG reagents (Invitrogen), according to the manufacturer's specification, with the cDNA equivalent of 10 ng of RNA in a 25 µl reaction volume. Reactions were performed in triplicate, and the absence of genomic DNA and primer dimers was confirmed by analysis of RT-minus and water control samples, and by examination of dissociation curves. Analysis of the amplification curves with LinReg software (Ramakers et al., 2003) showed that the efficiency of each PCR primer pair was above 0.95. Relative quantities were calculated with qBase v1.3.4 software (http://medgen.ugent.be/qbase/), taking primer efficiencies into consideration. Virtually identical results were obtained when using UBC or 18S rRNA as the reference for normalization across samples. For the whole sample set, a biological repeat yielded similar results.
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Western blotting
Seedlings were grown for 3 d in 30 µmol m–2 s–1 of blue light (470 nm) from Dragon Tape LEDs (Osram), harvested, and frozen in liquid nitrogen. Frozen tissue was ground with a 5-mm-diameter stainless-steel ball in a 2 ml Eppendorf tube using a ball mill (model MM301; Retsch, Haan, Germany). Boiling loading buffer (Laemmli) was added to the frozen ground tissue. The sample was then kept at 100 °C for 10 min and spun briefly to remove cell debris. Separation of proteins was carried out by SDS electrophoresis on a 12% polyacrylamide gel. Coomassie staining was done for 18 h, followed by appropriate destaining in 20% methanol, 10% acetic acid.
Blotting on nitrocellulose membrane was performed in an EBU-202 electrophoretic blotting system (C.B.S., Del Mar, CA, USA). The blot was blocked for 2 h in PBS with 1% dried skimmed milk (with vitamins; Nestlé) and the membrane was washed three times for 15 min in PBS. Hybridization with anti-GFP antibody (dilution 1:3000) was in PBS-T 1% skimmed milk for 6 h. The blot was washed once for 15 min with PBS, hybridized with peroxidase-linked anti-rabbit antibody (Sigma) in PBS for 2 h, and washed again in PBS-T twice for 10 min. Finally, the blot was developed using an enhanced luminol kit (Perkin Elmer) and exposed to Kodak X-omat film. A band with a molecular weight greater than 80 kDa was recognized as the DELLA–GFP fusion protein.
Epifluorescence microscopy
Seedlings were harvested and analysed within 15 min after harvest. A Zeiss Axioplan (Zeiss, Göttingen, Germany) inverted microscope with fluorescent light source and coupled to an Apotome was used. Signal intensity was determined using Zeiss Axiovision 4.6.
GUS staining
Seedlings were harvested and submerged in ice-cold 90% acetone for 30 min. They were then washed in 1 M phosphate, submerged in staining solution, and incubated for 6 h at 37 °C or for 18 h at room temperature. Staining solution contained 0.1 M phosphate buffer pH 7.2, 0.5 mM K ferricyanide, 0.5 mM K ferrocyanide, and 2 mM X-Gluc. Subsequent chlorophyll removal was done by keeping seedlings in 75% ethanol at 4 °C.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDISCUSSIONSupplementary dataReferences |
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Blue-light dependence of ethylene-regulated hypocotyl elongation
In long-day conditions in cool white light, ethylene or its precursor ACC stimulate hypocotyl elongation in Arabidopsis, especially on a low-nutrient medium (Smalle et al., 1997; De Paepe et al., 2005). To investigate this process in more detail, the conditions were simplified by using continuous light, and different fluence rates and spectral qualities were tested (Fig. 1). Seedlings were grown in the indicated light conditions for 6 d.
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Since red and blue light are the components of cool white light with demonstrated roles in hormonal control of stem growth, the effects of these wavelengths were compared on hypocotyl elongation at different fluence rates in wild type and ethylene mutants (Fig. 1a, b). In red light, which is less effective than blue light in inhibiting hypocotyl elongation (Kranz, 1977), the hypocotyl of the ctr1-1 mutant was slightly but significantly (t-test: P <0.01) smaller than that of the wild type, whereas in blue light at fluence rates above 10 µmol m–2 s–1 the ctr1-1 hypocotyl was significantly longer than that of the wild type. The ethylene-insensitive mutants or wild type treated with the ethylene action blocker 2,5-norbornadiene did not differ from untreated wild type in high-intensity blue light, indicating that endogenous ethylene production is limited in these conditions (Fig. 1b, Supplementary figure 1 available in Supplementary data at JXB online). These data indicate that inhibition of hypocotyl elongation, as occurring under high intensities of blue light, but not in red light, is necessary for the ethylene elongation response to occur.
The main photoreceptors that confer blue light-mediated inhibition of hypocotyl elongation are the cryptochromes, CRY1 and CRY2 (Ahmad et al., 2002). The cry1cry2 double mutant grown in blue light reacted as dark-grown wild-type seedlings upon ACC treatment, with a reduction in hypocotyl length, illustrating the importance of the cryptochrome signalling pathway for the ethylene response to occur in blue light (Fig. 1c; Bleecker et al., 1988). These data suggest that the ethylene elongation response is only visible when hypocotyls are strongly inhibited in growth due to light signalling, whereas defects in photomorphogenesis lead to inhibition of hypocotyl elongation upon ethylene treatment as seen in the triple response in the dark (Bleecker et al., 1988). Double mutants in the blue light phototropin receptors phot1phot2 reacted as wild type (Col-0) to ACC treatment in blue light, indicating that the ethylene response is independent of phototropin action (Fig. 1c). Previous studies have shown that cryptochromes are dependent upon phytochromes to produce their responses in blue light (Ahmad and Cashmore, 1997): phyA mutants have an elongated hypocotyl in blue light (Neff and Chory, 1998). Moreover, the major growth-suppressing cryptochrome CRY1 physically interacts with PHYA (Ahmad et al., 1998), suggesting a co-action of both photoreceptors. Therefore, the ethylene effects on phyA and phyB mutants in blue light were also tested (Fig. 1c). The phyB-1 mutant reacted as wild type (Ler) when treated with ACC. As expected, the untreated phyA-211 mutant had a longer hypocotyl than wild type (Col-0) in blue light, but the stimulation of elongation induced by exogenous ACC was very similar to that of the wild type.
Regulation of GA biosynthesis by ethylene and cryptochromes
Blue light, probably acting through cryptochromes, negatively affects GA biosynthesis and/or response (Reid et al., 2002; Folta et al., 2003; Foo et al., 2006). The response was therefore tested of the wild type and cryptochrome mutants to ACC when grown in the presence of the ent-kaurene oxidase inhibitor paclobutrazol (PAC), which confers GA deficiency. Reversibility of the inhibitory effect of PAC on hypocotyl elongation by GA treatment had been previously demonstrated for wild-type Arabidopsis (Saibo et al., 2003). Both the wild-type response to ACC and the long hypocotyl in the untreated cry1cry2 double mutant were abolished by PAC in blue light (Fig. 1d), indicating that GAs are required for the ethylene-stimulated hypocotyl growth in blue light and confirming that GA biosynthesis and/or signalling is diminished by cryptochrome action (Folta et al., 2003).
A possible role for GA biosynthesis in stimulation of hypocotyl elongation by ethylene in blue light was investigated further by determining the effect of ACC treatment on expression of GA-biosynthetic genes in whole seedlings using real-time RT-PCR. Seedlings were harvested after 3 d of growth in 30 µmol m–2 s–1 of blue light. In cry1cry2 seedlings, ACC treatment resulted in lower transcript levels of the gene encoding the first enzyme in the GA biosynthesis pathway, ent-copalyl diphosphate synthase (CPS), whereas in wild-type plants, transcript levels of CPS were only slightly higher after ACC treatment (P=0.12) (Fig. 2), despite an increase in CPS transcription in the hypocotyl in these conditions, as visualized after GUS staining in pCPS::GUS lines (Supplementary figure 2 available in Supplementary data at JXB online). This suggests a post-transcriptional control of RNA levels.
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The expression of GA20ox1 and -2 and GA3ox1 and -2 was also examined, as these genes are positive regulators of GA levels that act towards the end of the GA biosynthesis pathways and are known to function in vegetative tissues (Coles et al., 1999; Mitchum et al., 2006; I Rieu, A Phillips, P Hedden, unpublished results). The GA 2-oxidase genes GA2ox1, -2, and -7 encoding GA-inactivating enzymes (Thomas et al., 1999) were also analysed, based on their high expression level (GA2ox2) or their response to blue-light treatment in publicly available microarray data sets (GA2ox1 and GA2ox7; http://web.uni-frankfurt.de/fb15/botanik/mcb/AFGN/atgenex.htm). GA20ox1 and GA3ox1 transcript levels were increased in the light by ACC treatment (Fig. 2b, d). By contrast, GA20ox2 expression was not affected by ACC treatment, while GA3ox2 expression decreased after this treatment (Fig. 2c, e). Expression of GA2ox1 and GA2ox7 genes was substantially reduced by ACC treatment in the light, whereas GA2ox2 expression was unaffected by ACC treatment. Transcript abundance for GA2ox1 and GA2ox7 was also severely reduced in the cry1cry2 mutant indicating that expression of these genes is promoted by cryptochrome signalling (Fig. 2f–h).
In summary, ACC treatment in blue light resulted in changes in steady-state transcript levels of several genes (GA20ox1, GA3ox1, GA2ox1, GA2ox7) that might indicate stimulation of GA biosynthesis, whereas the opposite effect was found for GA3ox2.
Ethylene signalling results in reduced levels of bioactive GA
In order to determine whether there is an effect of ethylene signalling on GA content, endogenous GA levels were measured in wild-type seedlings grown in blue light, in the presence or absence of ACC, as well as in the ethylene-insensitive mutant etr1-1 and the constitutive ethylene response mutant ctr1-1 (Fig. 3). GA24 levels were similar in all circumstances, with the exception of ctr1-1 as compared with the wild type. By contrast, GA9 levels were reduced in ctr1-1, whereas they were elevated in etr1-1, despite the fact that no clear effect of ACC was visible in the wild type (Fig. 3b). However, treatment with ACC caused reduced levels of the bio-active GA4 (Fig. 3c). This reduction in bio-active GA4 content as a result of increased ethylene signalling was supported by the low GA4 concentration in ctr1-1 mutants, and a GA4 content in etr1-1 mutants equal to or even higher than that of wild-type seedlings. Interestingly, the inactive metabolite of GA4, GA34, had the same pattern as GA4 (Fig. 3d). Together these data suggest that ethylene has a severe negative effect on GA 3-oxidase activity, thus resulting in lower levels of bio-active GAs.
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Regulation of DELLA protein accumulation by ethylene
Gibberellins exert their activity by promoting breakdown of DELLA proteins, presumed transcriptional regulators that are potent inhibitors of elongation growth. This has been clearly demonstrated for the Arabidopsis RGA protein (Fleck and Harberd, 2002; Achard et al., 2003; Fu and Harberd, 2003). Moreover, DELLA proteins may act as nodes in the interplay with other hormones. For instance, RGA is destabilized by GA in Arabidopsis roots, and ACC appears to delay this effect (Achard et al., 2003), consistent with respective enhancement and inhibition of root elongation. A similar interaction between GA and ethylene pathways was found in apical hook maintenance (Achard et al., 2003; Vriezen et al., 2004). Hence it is conceivable that, in the hypocotyl, a stimulation of elongation by ethylene in the light results from an alteration in the expression of genes for GA signalling components and/or the accumulation of their respective products. To address this question, seedlings grown in blue light at a fluence rate of 30 µmol m–2 s–1 for 3 d were tested for differences in transcript levels of GA signalling genes by semi-quantitative RT-PCR (Fig. 4a). The genes for the GA receptors and the DELLA proteins GAI and RGA showed no significant or reproducible differences in steady-state transcript level when plants were treated with blue light and/or ACC (Fig. 4a). Therefore, effects on DELLA protein accumulation by blue light and ethylene were investigated using plants containing a pRGA::GFP-RGA construct (Silverstone et al., 2001). Accumulation of GFP–RGA fusion was detected using epi-fluorescence in live material. The seedlings were grown in blue light, in the presence or absence of ACC. GFP–RGA protein accumulated to higher levels in ACC-treated wild-type seedlings (Fig. 4b, Supplementary figure 3 available in Supplementary data at JXB online). The enhancement of GFP–RGA accumulation by ACC treatment was also pronounced in the cry1cry2 mutant. This suggests that ethylene inhibits GA responses in this tissue. However, the accumulation of GAI remained unaffected by ACC both in blue light and in darkness (Fig. S4a available in Supplementary data at JXB online).
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The gibberellin signal in the hypocotyl is repressed by ethylene
The GA- inducible reporter gene pGASA1:: LUCIFERASE-GUS (3GLG) (Raventos et al., 2000) was used to evaluate the effect on GA-regulated downstream components. GUS staining was visible in the hypocotyl of untreated plants grown in blue light. Except for the root–shoot junction, this staining was absent in the hypocotyl of ACC-treated seedlings, suggesting reduced GA signalling in the cells involved in hypocotyl elongation (Fig. 5; Le et al., 2005).
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Ethylene-stimulated hypocotyl elongation in blue light is facilitated by, but does not act through, an increase in GA signalling
To investigate further whether GAs are involved in ethylene-stimulated hypocotyl elongation, the ethylene responsiveness of GA biosynthesis and signalling mutants was tested. Seedlings were grown in 30 µmol m–2 s–1 blue light for 6 d. ga1-1 and ga1-3 plants, which have very low GA levels, showed a very limited response, far less than other GA mutants, indicating that a minimal level of GA is needed for the ethylene-induced elongation response to occur (Fig. 6a). In accordance with this, a limited response to ACC was also observed in a dwarfed GAI overexpresser line (Supplementary figure 4b available in Supplementary data at JXB online), which is consistent with the weak response of gai gain-of-function mutants in white light (Vriezen et al., 2003a). The ga20ox1-1 (ga5-1) mutant still gave a clear response to ACC, although less strong than the wild type. Despite the difference in transcript accumulation of GA2ox1 and GA2ox7 in seedlings grown on ACC (Fig. 2), loss-of-function mutants of these genes reacted as the wild type to the treatment, with an increase in hypocotyl length of 1.5- to 2-fold (Fig. 6a), consistent with GAs not being the factor controlled by ethylene during stimulation of hypocotyl growth.
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Combined DELLA loss-of-function mutants (rga24gai-t6 and rga24gai-t6rgl1rgl2; Achard et al., 2006) had a longer hypocotyl than the wild type, as expected given their constitutive activation of GA responses. Hypocotyl length in the DELLA quadruple loss-of-function mutant is similar to that obtained by treating the wild type with GA4, and treatment with GA4 did not stimulate hypocotyl growth further (Fig. 6b), suggesting that these four DELLA proteins are sufficient to mediate inhibition of elongation growth in the hypocotyl via suppression of the GA signalling pathway. However, the DELLA quadruple knock-out, even when treated with GA4, still responded vigorously to ACC. Moreover, while exogenous GA4 increased hypocotyl length in the wild type to the levels reached upon ACC treatment, the combination with ACC induced extra elongation (Fig. 6b).
When PAC was used to block GA biosynthesis, wild-type seedlings treated with ACC showed only a very limited hypocotyl elongation (Fig. 6c), which is in accordance with the results for ga1 mutants (Fig. 6a), indicating that a basal GA level is necessary for the ethylene response to occur. Hence, the ethylene response is GA dependent, basal levels of GA being necessary (Fig. 6a), yet not mediated by GA, since DELLA knock-out mutants had a clear response (Fig. 6b). Hypocotyl length in rga24gai-t6rgl1rgl2 seedlings was not reduced by PAC treatment, and there was a clear hypocotyl elongation response when PAC and ACC treatments were combined (Fig. 6c). These data again suggest that GA-controlled hypocotyl elongation is fully dependent on these four DELLA proteins, whereas the ethylene-stimulated elongation is at least partly independent of these.
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DISCUSSION |
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TopAbstractIntroductionMaterials and methodsResultsDISCUSSIONSupplementary dataReferences |
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Ethylene and regulation of GA biosynthesis
Both ethylene and GA have similar stimulatory effects on hypocotyl elongation in the light (Peng and Harberd, 1997; Smalle et al., 1997). Therefore, a possible ethylene control of GA signalling pathways was investigated. Unexpectedly, treatment of Arabidopsis seedlings with the ethylene precursor ACC in the light resulted in a reduction in the concentration of GA4, the main bio-active GA in Arabidopsis (Fig. 3). This coincided with a reduction in transcript levels for GA3ox2 (Fig. 2), which encodes a GA4-forming enzyme, and with an accumulation of the GA-regulated growth repressor DELLA protein RGA (Fig. 4). However, these indications of a low GA signal coincided with higher levels of CPS, GA20ox1, and GA3ox1 transcripts of GA biosynthesis genes and lower levels of GA2ox1 and GA2ox7 transcripts of GA-inactivating genes (Figs 2, 3). A similar finding for GA20ox1 and GA3ox1 transcripts was found at later stages in development in the presence of constitutive ethylene signalling, suggesting a more general effect of ethylene signalling on GA biosynthesis (Achard et al., 2007b). Apart from the effect on CPS expression, these changes in gene expression are suggestive of the feedback pathway that operates in GA metabolism, in which reduced active GA levels result in DELLA protein accumulation, causing an increase in transcript levels of GA biosynthetic genes and down-regulation of genes encoding GA-inactivating enzymes (Phillips et al., 1995; Cowling et al., 1998; Ait-Ali et al., 1999; Xu et al., 1999). The CPS gene is not feedback regulated, as treatment with the GA biosynthesis inhibitor PAC does not alter its expression pattern (Vriezen et al., 2004). CPS expression may therefore be directly regulated by ethylene (Fig. 2; Vriezen et al., 2004). Thus, it is proposed that the main ethylene action on GA signalling is by stabilizing DELLA proteins through reduction of bio-active GA levels (Fig. 7). It is speculated that, in view of the discrepancy between transcript regulation of most of the GA 3-oxidase and GA 2-oxidase genes and GA4 levels (Figs 2c, d, 3c
), enzyme activity may be regulated at the post-transcriptional level. The difference in accumulation patterns of GA9, a product of GA 20-oxidases, and GA24, both a product of and substrate for these enzymes, in ACC-treated wild-type, ctr1-1, and etr1-1 seedlings (compare Fig. 3a and b) indicates that GA 20-oxidase activity may also be affected by ethylene signalling, either directly or indirectly.
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Cryptochromes, ethylene, and GA signalling
Cryptochromes can regulate GA biosynthesis at the level of transcript accumulation (Fig. 2), consistent with earlier published data (Folta et al., 2003; Foo et al., 2006). Especially the GA-inactivating GA 2-oxidases had lower transcript levels in cry1cry2 mutant seedlings (Fig. 2e–g). Lack of cryptochromes in blue light thus leads to a situation similar to that in darkness, where photomorphogenic inhibition of hypocotyl elongation is not occurring. The shorter hypocotyl in darkness in the presence of ethylene is part of the triple response (Bleecker et al., 1988).
Cryptochromes are not the only photoreceptors that mediate inhibition of hypocotyl elongation in blue light. It is known that phytochromes absorb blue light to some extent and that phytochrome A mutants have a long hypocotyl phenotype in blue light (Zhou et al., 2005). In addition, cryptochromes and phytochromes can co-activate photomorphogenesis (Neff and Chory, 1998), and PHYA and CRY1 were shown to interact physically (Ahmad et al., 1998). Since ACC still stimulates hypocotyl elongation of phyA mutants in blue light (Fig. 1e), the ethylene signal probably does not act on the photoreceptors but on a downstream component, common to far-red and blue light signalling, or affects only the cryptochrome-dependent pathways in blue light.
Independent but co-acting hormonal pathways for control of hypocotyl elongation
Blue light, through cryptochrome signalling, inhibits elongation as part of the photomorphogenesis programme (Ahmad et al., 2002). This pathway represses GA, auxin, and brassinosteroid signals (Ma et al., 2001; Folta et al., 2003). Ethylene and its precursor ACC, on the other hand, stimulate hypocotyl elongation in blue light (Fig. 1; Smalle et al., 1997). The GA20ox1 and GA3ox1 genes are important determinants of GA-dependent hypocotyl growth, as corresponding loss-of-function mutations cause semi-dwarfism, while other members of the GA20ox and GA3ox gene families have not been identified in screens for GA-dependent dwarfism (Koornneef and van der Veen, 1980; Coles et al., 1999; Mitchum et al., 2006). In addition, only a slight stimulation of hypocotyl growth was seen upon ACC treatment of the ga5-1 (GA20ox1) loss-of-function mutant in blue light (Fig. 6a). Therefore, increases of transcript levels of GA20ox1 and GA3ox1 might have been expected to confer extra elongation due to higher GA levels and signal (Huang et al., 1998; Coles et al., 1999; Radi et al., 2006). However, ethylene does not seem to promote growth through stimulation of the GA signal, as ACC treatment results in lower levels of bio-active GA4 and accumulation of the RGA DELLA protein, a negative regulator of GA responses. This is another example of enhanced elongation without concomitant reduction of DELLA protein levels. Recently, it has been demonstrated that spindly-8 (spy-8) which partially suppresses all phenotypes of the dominant GA-insensitive dwarf mutant rga-
17, does not reduce rga-17 or RGA protein levels (Silverstone et al., 2007). The present results might therefore indicate a mechanism of ethylene regulation of SPY function. The DELLA accumulation is consistent with the reduction in expression of GASA1, a positive marker for GA signalling, in the hypocotyl upon ACC treatment in blue light. On the other hand, ACC produces little stimulation of elongation in PAC-treated wild type or in GA biosynthesis and signalling mutants with high DELLA levels, indicating their role as limiting factors, whereas seedlings having a constitutive GA response (quadruple DELLA loss-of-function mutants) respond strongly to ACC. Therefore, the elongation response to ethylene in the light is not mediated by GA pathway(s), although, paradoxically, the presence of an active GA signalling pathway is required in a minimal, permissive level (Fig. 7). The ethylene response is thus dependent on GA, yet not mediated by GA. Consequently, the two pathways must function for ethylene-induced hypocotyl elongation to occur: GA signalling appears to facilitate the ethylene response although acting separately from it. Recently it was demonstrated that GA partly regulates gene expression independent of DELLA protein function (Cao et al., 2006). Thus, the possibility exists that part of the hypocotyl elongation response controlled by GA is independent of DELLA proteins. However, the present data indicate that this is not the case, because GA-regulated hypocotyl elongation appears completely mediated through the DELLA proteins (Fig. 6b, c).
At this point, the mechanism for the ethylene response is unknown, but there are clear indications that auxin and brassinosteroid signalling may be involved downstream of ethylene (Saibo et al., 2003; Vandenbussche et al., 2003b; De Grauwe et al., 2005).
In conclusion, it has been shown that the blue-light inhibition of hypocotyl elongation is counteracted by ethylene treatment independently of a stimulation of a DELLA-mediated GA signalling pathway (modelled in Fig. 7). Indeed, ethylene seems to inhibit the GA signal by causing lower GA levels and DELLA protein accumulation. The presence of high levels of DELLA proteins is not consistent with the observed growth response and suggests that ethylene must act on one or more alternative pathways, most probably involving auxin and brassinosteroids (De Grauwe et al., 2005), that act independently of DELLA proteins and can over-ride their action. However, the ethylene-regulated pathway is dependent on the remaining (low) GA signal which is necessary for elongation. Whether ethylene acts by direct stimulation or by derepressing the inhibition of elongation by light remains to be determined (Fig. 7). Future research involving an in-depth analysis of these pathways will further unravel the complexity of hormonal control of Arabidopsis hypocotyl elongation in the light. This may also provide a mechanism for ethylene-stimulated growth that is different from those observed in other species, such as Rumex, Ranunculus, and deep-water rice (Vriezen et al., 2003b; Pierik et al., 2006).
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResultsDISCUSSIONSupplementary dataReferences |
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Supplementary figure 1. Effect of 2.5-norbornadiene (NBD) on hypocotyl elongation in blue light.
Supplementary figure 2. Analysis of a promoter-GUS reporter construct of the ent-copalyl diphosphate synthase gene (CPS; GA1) (Silverstone et al., 1997) in wild type or cry1cry2 background.
Supplementary figure 3. Signal intensities of figure 4b.
Supplementary figure 4. Effect of ACC on a 35S::GAI-GFP overexpressor line. (a) Western blot of 35S::GAI-GFP seedlings, grown for 3 days in blue light, with or without 50 µM exogenous ACC. (b) Hypocotyl lengths of the distinct segregating phenotypic classes in the offspring of a heterozygous 35S::GAI-GFP parental line.
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Acknowledgements |
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FV is a post-doctoral fellow from the Research Foundation – Flanders (FWO). This work was funded by grants from the Research Foundation – Flanders (FWO) (G.0345.02, G.0313.05) to DVDS and Krediet aan Navorsers (1.5.042.0 [EC] 6) to FV. Rothamsted Research is sponsored by the Biotechnology & Biological Sciences Research Council of the UK, which also supported IR through grant P19317 [GenBank] .
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