JXB Advance Access originally published online on January 10, 2005
Journal of Experimental Botany 2005 56(413):841-849; doi:10.1093/jxb/eri078
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RESEARCH PAPER |
RP-ACS1, a flooding-induced 1-aminocyclopropane-1-carboxylate synthase gene of Rumex palustris, is involved in rhythmic ethylene production
1Department of Experimental Botany, Radboud University Nijmegen, Toernooiveld 1, 6525 ED, Nijmegen, The Netherlands
2Department of Molecular and Laser Physics, Radboud University Nijmegen, Toernooiveld 1, 6525 ED, Nijmegen, The Netherlands
3Department of Plant Ecophysiology, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands
To whom correspondence should be addressed. Fax: +31 24 3652490. E-mail: w.vriezen{at}science.ru.nl
Received 27 July 2004; Accepted 9 November 2004
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Abstract |
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Many semi-aquatic plants respond to flooding by elongating the shoot to reach the water surface. This response is initiated by accumulation of ethylene in the plant due to decreased gas-exchange and continued ethylene production during submergence. Ethylene biosynthesis is often limited by the availability of 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, synthesized by ACC synthase. Here, is reported the cloning of a Rumex palustris cDNA corresponding to an ACC synthase gene (RP-ACS1), whose expression is induced by submergence in the long term but does not precede the observed short-term increase in ACS activity. Under aerated conditions, RP-ACS1 messenger accumulation exhibited circadian rhythmicity with high levels in the dark phase and low levels in the light phase, similar to the oscillations in ethylene production under these conditions. ACC oxidase (RP-ACO1) messenger accumulation also showed a rhythmic pattern, but opposite to that of RP-ACS1, and closely resembled the ethylene oscillation found in R. palustris plants that were waterlogged. Together the results indicate that transcriptional regulation of RP-ACS1 may directly control rhythmic ethylene production under aerated condition and suggest that post-transcriptional regulation is important in initial up-regulation of ACS activity upon submergence.
Key words: Ethylene, ACC synthase, submergence, Rumex, circadian rhythm
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Introduction |
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The gaseous plant hormone ethylene has been shown to influence many processes in plants such as germination, root-hair initiation, flower senescence, and fruit ripening. Furthermore, responses to a wide variety of stresses such as drought, wounding, pathogen attack, and waterlogging depend on ethylene and are initiated by an increase in ethylene biosynthesis (Jackson, 1985
; Abeles et al., 1992). The ethylene biosynthetic pathway has been extensively studied in many plant species (Yang and Hoffman, 1984; Kende, 1993; Fluhr and Mattoo, 1996). The precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), is produced via conversion of S-adenosylmethionine (SAM) by ACC synthase, and ACC is converted to ethylene, CO2, and HCN by ACC oxidase in an O2-dependent process. In a number of plants ACC synthase and ACC oxidase proteins are encoded by gene families whose members are differentially regulated (Fluhr and Mattoo, 1996). Generally, the rate-limiting step in ethylene biosynthesis is the conversion of SAM to ACC (Yang and Hoffman, 1984), and an increase in ethylene production is often preceded by induction of ACC synthase genes (Rottmann et al., 1991; Botella et al., 1993; Oetiker et al., 1997), although post-transcriptional regulation of ACC synthase has also been observed in some cases (Spanu et al., 1994; Vogel et al., 1998). ACC oxidase acitvity can be limiting in ethylene biosynthesis in situations of high ethylene production or low oxygen concentration (English et al., 1995; Yamamoto et al., 1995; Barry et al., 1996; Lasserre et al., 1996; Vriezen et al., 1999).
Rumex palustris is a semi-aquatic plant that grows mainly in flooding-prone river areas, and serves as a model species for studying the physiology of flooding tolerance (Peeters et al., 2002; Voesenek et al., 2003a). Hyponastic growth and rapid elongation of the petiole cells are major processes which keep the foliage of R. palustris plants above the rising water surface (Voesenek et al., 1990b; Cox et al., 2003). Ethylene plays an important role in the induction and maintenance of this response, as it does in other semi-aquatic and amphibious species (Musgrave et al., 1972; Metraux and Kende, 1983; Blom et al., 1994). Furthermore, in R. palustris, enhanced rates of ethylene production are important for fast elongation to continue when shoot tips emerge from the floodwater (Voesenek et al., 2003b). It has been reported previously that, upon submergence, the conversion rate of ACC to ethylene decreases, although ACO transcript level and in vitro protein activity increase (Banga et al., 1996; Vriezen et al., 1999). The rate of conversion of SAM to ACC is temporarily induced by submergence, but the contributions of the ACS gene and the activity of the corresponding protein are unkown (Banga et al., 1996).
Under drained conditions, R. palustris produces ethylene at a low level. However, when the soil is saturated with water (waterlogging) ethylene biosynthesis is induced and the ethylene release shows a clear rhythm with peaks during the day (Voesenek et al., 1990a). Rhythmic ethylene production has been observed in various species (El-Beltagy et al., 1976; Rikin et al., 1984; Ievinsh and Kreicbergs, 1992; Kathiresan et al., 1996; Machácková et al., 1997; Beßler et al., 1998; Finlayson et al., 1998; Dziubinska et al., 2003; Yamasaki et al., 2003) and can have a circadian nature (Rikin et al., 1984; Finlayson et al., 1998; Dziubinska et al., 2003), or be under strict control of the light (Beßler et al., 1998). The conversion step at which rhythmic ethylene production is regulated also differs between species. As expected, in most species ACC synthesis seems to determine the formation rate of ethylene (Rikin et al., 1984; Machácková et al., 1997; Beßler et al., 1998; Yamasaki et al., 2003). However, fluctuations in ACC oxidase activity are responsible for generating the ethylene rhythm in an ethylene-overproducing cultivar of Sorghum bicolor (Finlayson et al., 1999) and may also be in Stellaria longipes, in which ACC oxidase activity oscillates in phase with ethylene production (Kathiresan et al., 1996).
To study the role of ACC synthase in ethylene production in R. palustris during submerged and non-submerged conditions, the most abundant R. palustris ACC synthase gene, RP-ACS1, was identified, and its expression compared with ACC synthase activity and ethylene production. Expression of RP-ACS1 was induced by submergence, but the induction did not coincide with induction of ACS enzyme activity. Under aerated conditions RP-ACS1 mRNA concentration fluctuated in a rhythm that was highly similar to the rhythm observed in ethylene production.
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Materials and methods |
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Plant material and growth conditions
Achenes of Rumex palustris Sm. were collected from river areas near Millingen, The Netherlands. Germination and growing conditions were as described by Banga et al. (1996)
. All of the plants used for experiments were 26–30-d-old. Plants were grown under alternating light regimes (16 h light; PPFD 65 µmol m–2 s–1; 8 h dark) at a constant temperature of 22 °C. For the experiments involving submergence, plants were kept under constant light and temperature, starting 24 h before submergence. Submergence took place in an open tank with 25 cm tap water at 22 °C. At several time points after submergence shoots or petioles and lamina of leaf 4, the youngest fully developed leaf, were cut and directly frozen in liquid nitrogen. Northern analysis showed that the expression patterns of the genes studied were comparable in leaves 3, 4, and 5 of 4-week-old R. palustris plants (data not shown). For the experiments under aerated conditions, the alternating light regime was continued for two full cycles and then the regime was changed to constant light, all at constant temperature.
Isolation of ACC synthase cDNA fragments
Primers TZ-1F
and TZ-2R, which were based on conserved ACC synthase protein sequences, have been described before (Zarembinski and Theologis, 1993). Degenerated primers RP-SYN2 and RP-SYN3 were based on conserved DNA sequences of known ACC synthase sequences obtained from GenBank (RP-SYN2: 5'-CCCAKCRGCYTCAATYTGYAC-3'; RP-SYN3: 5'-CCRAYTCKRAADCCWGGBARSCCCAT-3' using IUB codes). cDNA was isolated from an R. palustris cDNA Uni-ZAP XR library (Vriezen et al., 1997) using the mass excision protocol of the manufacturer (Stratagene). The isolated pBluescript phagemid was used for PCR reactions after phenol–chloroform extraction and ethanol precipitation. A 200 ng aliquot of this DNA was used for a PCR reaction in 100 µl PCR buffer with 1.25 mM MgCl2, 200 pmol of each dNTP, 100 pmol of each primer RP-SYN2 and RP-SYN3, and 0.8 units of thermostable DNA polymerase (Goldstar, Eurogentec). Thirty cycles of 1.5 min at 94 °C, 1 min at 54 °C, and 1 min at 72 °C were performed, preceded by 4 min at 96 °C, and terminated with 10 min at 72 °C, in a Thermal Cycler (Perkin Elmer).
A second PCR reaction was performed with 200 ng of the pBluescript phagemid in 100 µl PCR buffer with 1.5 mM MgCl2, 200 pmol of each dNTP, 100 pmol of each primer TZ-1F and TZ-2R, and 0.4 units of thermostable DNA polymerase. The amplification reaction consisted of five cycles of 1.5 min at 94 °C, 1 min at 37 °C, and 1 min at 72 °C, followed by 35 cycles of 1.5 min at 94 °C, 1 min at 45 °C, and 1 min at 72 °C, preceded by 4 min at 96 °C, and terminated with 10 min at 72 °C. A 5 µl aliquot of this reaction mixture was re-amplified under the same conditions for 35 cycles. After the DNA fragment size was verified, the PCR products (a 152 bp fragment with the TZ-1F and TZ-2R primers and a 660 bp fragment with the RP-SYN2 and RP-SYN3 primers) were cloned into the pCRII vector using a TA Cloning Kit (Invitrogen) and sequenced.
cDNA library screening
Using a R. palustris cDNA library (Vriezen et al., 1997) approximately 0.8x106 plaques were screened with a [-32P]dATP-labelled 660 bp ACC synthase PCR fragment according to the manufacturer's protocol (Statagene). Filters were prehybridized for 1 h and hybridized overnight at 65 °C with a solution containing 5xSSC, 5xDenhardt's reagent, 0.5% (w/v) SDS, and 100 µg ml–1 denatured, fragmented salmon sperm DNA. Membranes were then washed twice in 2xSSC plus 0.1% (w/v) SDS at 65 °C for 15 min each and twice in 0.2xSSC plus 0.1% (w/v) SDS for 15 min. The blots were exposed to film (X-Omat AR, Kodak) with two intensifying screens at –80 °C for 16–48 h. After three rounds of screenings 13 positive plaques were identified and 10 were sequenced and proved to be identical ACC synthase homologues (RP-ACS1; Genbank accession number AF038945).
DNA and RNA isolation and blot hybridization analysis
Genomic DNA was isolated according to Van Eldik et al. (1995). Southern blotting, DNA fixation, and hybridization were performed on a nylon membrane (Hybond-N, Amersham) according to the manufacturer's directions. The full-length RP-ACS1 cDNA and the pcr7Rp fragment were labelled with [-32P]dATP by the random-priming method, and used as probes. Prehybridization, hybridization, and washing conditions were the same as described for the library screening.
For RNA gel blots, total RNA was isolated according to Van Eldik et al. (1995) and separated on a 1% (w/v) agarose gel containing 0.4 M formaldehyde and 0.1 µg ml–1 ethidium bromide. RNA was transferred to a nylon membrane (Hybond-N, Amersham) according to the manufacturer's directions. Prehybridization, hybridization, and washing conditions were the same as described for the library screening. Hybridizations were performed using the full-length cDNAs RP-ACS1, RP-ACO1 (accession no. Y10034), RP-CAB1 (accession no. AF165529), or Rp-EXP1 (accession no. AF167360) as probes. The blots were exposed to film (X-Omat AR, Kodak) with two intensifying screens at –80 °C for 10–14 d (RP-ACS1 and Rp-EXP1) or 1–2 d (RP-ACO1 and RP-CAB1). For hybridization with the RP-ACO1 probe, the blots used for RP-ACS1 hybridization were stripped and rehybridized with labelled RP-ACO1, which was later repeated for Rp-EXP1. Finally, hybridization with a tobacco ribosomal cDNA (kindly provided by Dr K Weterings, University of Nijmegen) was performed to confirm equal loading of the membranes. The autoradiographs were scanned with a densitometer (Molecular Imager FX, Bio-Rad), and the signals were quantified (Molecular Analyst, Bio-Rad). The value for the messenger concentration is the density of the band on the autoradiograph corrected for the density of the 28S band. All analyses of messenger concentrations were done at least twice with different plants in different experiments to ensure that the observed patterns of the mRNA concentration were reproducible.
Analyses of in vitro ACC synthase capacity
Shoots were excised at the soil surface, weighed, frozen in liquid N2 and subsequently stored at –80 °C. Prior to extraction, samples were ground in liquid N2. Half a millilitre of extraction buffer (600 mM TES pH 8.5, 5 mM DTT, 10 µM pyridoxal-5-phosphate; pH adjusted using KOH) was added per gram fresh weight. The slurry was centrifuged at 20 000 g for 20 min at 4 °C and the supernatant dialysed overnight against two changes of buffer (10 mM TES pH 8.5, 10 µM pyridoxal-5-phosphate) at 4 °C. A minimum of 25 ml of incubation dialysis buffer was used per millilitre of supernatant. In the assay, 150 µl extraction buffer was added to 1 ml supernatant and 100 µl AdoMet solution (10 mM). The vial was closed air-tight with a septum and the reaction mixture was placed on an orbital shaker at 30 °C for 1 h. ACC was determined by the method of Lizada and Yang (1979) with internal standardization (ACC from Sigma). ACC was chemically converted into ethylene, which was measured with a gas chromatograph [Synspec GC 955-100 equipped with a photo ionization detector and a stainless-steel column (0.12x2.0 m) filled with Haysep R mesh 80/100 (temperature 105 °C; carrier gas N2)]. This experiment was repeated with similar results. To test for possible differences in the recovery of ACC synthase activity from tissues isolated under control conditions (low expected activity) and after 2 h of de-submergence (high expected activity), equal masses of tissue homogenates were mixed and the ACC synthase activity was determined. The activity varied between 2% and 4% of the expected activity calculated for the mixed samples.
Ethylene measurements
For ethylene measurements, plants were transferred from a tray to a Petri dish (two plants per Petri dish) after gently removing the surplus of soil, which was not in contact with the roots. The Petri dishes were placed in cuvettes and the open cuvettes were kept in the growth chamber used for ethylene measurements for 2 d to acclimatize the plants. Some of the removed soil was put in a Petri dish to serve as a control in the measurements. Plants were kept under alternating light regimes (16 h light; PPFD 150 µmol m–2 s–1; 8 h dark) and later in the experiment under continuous light, always at a constant temperature of 21 °C. Plants were watered well to prevent closure of the stomata in the light. Ethylene measurement was performed using a sensitive laser-based photoacoustic detector in combination with a gas flow-through system (flow of 2.5 l h–1), as described before (Montero et al., 2003 and references therein). Measurements were done twice, with three cuvettes in each measurement (two with plants and one with soil); representative data are shown in the Results.
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Results |
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Cloning of ACC synthase cDNAs from R. palustris
Partial cDNAs encoding ACC synthase were generated by PCR with degenerated primers on a cDNA library constructed from leaf RNA isolated from R. palustris plants submerged for 24 h. Two combinations of degenerated oligonucleotides were used for this reaction. The primer combinations TZ-1F
/TZ-2R and RP-SYN2/RP-SYN3 led to the synthesis of a 152 bp and a 660 bp fragment, respectively. Sequence analysis of several subclones of these fragments demonstrated that they were all 100% homologous in the overlapping sequences, indicating that they all derived from the same cDNA. The 660 bp fragment showed high homology to known ACC synthase sequences from other species and was subsequently used to isolate full-length clones from the same R. palustris cDNA library. Ten of the clones obtained proved to be identical in sequence, and the corresponding gene was designated RP-ACS1. The deduced RP-ACS1 protein consists of 489 amino acids and has a molecular weight of 55 kDa and a pI of 6.89. It contains all seven domains that are conserved in many ACC synthases (Dong et al., 1991; Theologis, 1992) and also the 11 amino acids conserved in various aminotransferases (Rottmann et al., 1991). The RP-ACS1 protein shares highest similarity (68%) with a functional ACC synthase from citrus (CS-ACS1; Wong et al., 2001).
Furthermore, sequencing of PCR fragments pcr2Rp and pcr7Rp (accession nos AF041480 and AF041481) amplified from R. palustris genomic DNA with primer combination TZ-1F/TZ-2R showed the existence of at least two additional ACC synthase genes. Southern analysis of genomic DNA using the full-length RP-ACS1 cDNA and pcr7Rp as probes confirmed the presence of a multi-gene ACC synthase family (Fig. 1). However, PCR with the same TZ-1F/TZ-2R primers on cDNA isolated from the cDNA library, and RNA gel-blot analyses of R. palustris RNA using the pcr2Rp or pcr7Rp fragment as a probe, did not produce evidence for the presence of messengers that could correspond to the other two genes, either under submerged or non-submerged conditions.
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RP-ACS1 expression upon submergence
RP-ACS1 messenger could be detected in total shoots (five petioles + five leaf blades) and in the roots of young R. palustris plants that were submerged and kept under constant light and temperature. Figure 2A shows that the RP-ACS1 mRNA level in the shoot remained practically constant during the 12 h of submergence, and started to accumulate to higher levels at 24 h that persisted up to 48 h. RP-ACS1 was also expressed in the roots during this treatment, but no increase was observed after submergence. By contrast, direct measurement of ACC synthase activity in shoots of submerged and non-submerged plants showed that activity of the protein was strongly enhanced after 6 h of submergence, after which it declined again (Fig. 2B). The plants used in all these experiments were composed of five leaves, arranged in a rosette, of which the youngest (leaves 4 and 5) are the most responsive to ethylene (Voesenek et al., 1990b
). To ensure that a local increase in RP-ACS1 transcript level in responsive tissue had not been overlooked, due to dilution with non-responsive tissue, RNA from petioles of leaf 4 was analysed separately. Figure 2C also shows that, in leaf 4, RP-ACS1 transcript level remained constant during the first 12 h after submergence. Surprisingly, however, analysis of RP-ACS1 transcript accumulation in leaf 4 of non-submerged control plants showed that during 1 d the transcript level decreased significantly.
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Further localization of the expression of RP-ACS1 was performed by analysing total RNA of several segments of the petioles and lamina of leaf 4. Figure 3 shows that before submergence (t=0 h) the majority of the ACC synthase mRNA was localized in the petioles and was low, but detectable, in the lamina. After 24 h of submergence the RP-ACS1 mRNA level had increased in both tissue types, but was still higher in the petioles.
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Rhythmic messenger accumulation
Modulation of RP-ACS1 mRNA levels in non-submerged plants (Fig. 2C) led us to analyse the expression pattern of the R. palustris ethylene biosynthetic genes in the shoot at several time points over 2 d with an 8 h dark phase and an additional 2.5 d with constant light (Fig. 4). The expression of chlorophyll a/b-binding protein (CAB) genes is known to show a strong circadian rhythm that persists under constant light (McClung, 2001
). The messenger level of a R. palustris CAB gene (RP-CAB1; unpublished results) was used as an endogenous control of the circadian rhythm during these experiments. The first sample every day was taken 30 min before the end of the dark phase. The RP-CAB1 messenger levels were still low at this time point but increased rapidly to a maximum in the middle of the light phase. The amplitude and the period of this rhythm were not affected by exposure to constant light for two nights (Fig. 4B). The messenger accumulation of RP-ACS1 also showed a rhythmic pattern, but opposite to that of RP-CAB1 (Fig. 4C). The highest levels were found when the plants were still in the dark, and the lowest levels were found at the middle of the light phase. Cycling of the ACC synthase transcript levels persisted for at least 2 d in the plants when subjected to continuous light, although the amplitude of the rhythm decreased strongly. The ACC oxidase (RP-ACO1, 1.3 kb; Vriezen et al., 1999) messenger accumulation displayed a rhythm with the peaks in the middle of the light phase as was found for the RP-CAB1 mRNA accumulation. However, the amplitude of the rhythm was drastically reduced after the cycle under constant light, and no increase in messenger was seen after the second cycle (Fig. 4D; days 4 and 5).
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Rhythmic ethylene production
Under non-submerged conditions, it is expected that ACC synthase activity is rate-limiting in ethylene production. To determine possible consequences of oscillations in RP-ACS1 transcript levels, measurement of ACC synthase activity and ethylene production of R. palustris plants grown under the same light regime was attempted. However, under non-submerged conditions ACC synthase activity was too low for accurate measurement (see control plants in Fig. 2B). Ethylene production was analysed using laser-driven photoacoustic spectroscopy, a system able to measure ethylene in the pl l–1 area (Montero et al., 2003
and references therein). Figure 5A shows that during the light–dark cycles ethylene production was low in the light phase and increased in the dark phase. Under constant light conditions this rhythm persisted, with lower amplitude. It has been reported that under light conditions photosynthesis may inhibit ethylene production by decreasing the level of carbon dioxide in the cuvette (Kao and Yang, 1982). Carbon dioxide levels in the cuvette were indeed lower in the light, and supplementing the incoming air with extra carbon dioxide resulted in an increase in ethylene production (data not shown). Although this effect may have enhanced the difference between ethylene production in the light and in the dark, the persistence of the rhythm in constant light indicated that ethylene production did oscillate. Rp-EXP1 encodes for an -expansin gene that is regulated by ethylene (Vriezen et al., 2000) and is used here as a marker for ethylene response. Figure 5B shows that its mRNA accumulates with a comparable pattern as RP-ACS1 mRNA (Fig. 4A, C), possibly following ethylene release (Fig. 5).
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Discussion |
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ACC synthase genes are members of gene families and have been shown to be induced or inhibited in different plant tissues by stimuli such as auxin (Zarembinski and Theologis, 1994
), wounding (Liu et al., 1993), submergence (Van der Straeten et al., 1997; Zarembinski and Theologis, 1997), and pollination (Bui and O'Neill, 1998). Also in R. palustris, multiple ACC synthase genes are present (Fig. 1). This suggests that the R. palustris ACC synthase genes may be differentially expressed and that more than one heterologous ACC synthase messenger may accumulate in a given tissue. However, using PCR with degenerated primers, it was possible to isolate only one ACC synthase cDNA (RP-ACS1) from leaves of R. palustris submerged for 24 h, although the same degenerated primers have been successfully used to amplify other ACC synthases from genomic DNA of R. palustris (pcr2Rp, pcr7Rp) and from R. acetosa, Nicotiana tabacum, Ranunculus sceleratus (data not shown), and rice (Zarembinski and Theologis, 1993). Because RNA gel blots probed with the ACC synthase gene fragments pcr2Rp and pcr7Rp also did not produce evidence for the presence of other messengers in R. palustris leaves under submerged or aerated conditions, it was assumed that RP-ACS1 is most likely to be the gene responsible for the major part of ACC production in the R. palustris tissues used in the experiments.
The role of RP-ACS1 during submergence
It has been reported that ACC concentration increases in the roots and shoots of submerged R. palustris plants (Banga et al., 1996) and also in the intercalary meristem of rice plants (Cohen and Kende, 1987; Zarembinski and Theologis, 1997), with the maximum levels reached within the first hour of submergence. Detailed studies on conversion rates and on ACC oxidase gene expression and activity have shown that ACC accumulation in R. palustris is caused, in part, by the decreased activity of ACC oxidase during submergence (Banga et al., 1996; Vriezen et al., 1999). In addition, Banga et al. (1996) calculated that submergence caused a short-term increase in ACC synthesis and this could be confirmed by measurement of ACC synthase activity (Fig. 2B). The present results show that RP-ACS1 transcript level in the root did not increase during 48 h of submergence and remained at a constant level for at least 12 h in the shoot (Fig. 2A, C). This discrepancy between RP-ACS1 messenger concentration and ACC synthesis suggests that ACC synthase activity is regulated at a post-transcriptional level, as had already been observed in other species (Spanu et al., 1994; Vogel et al., 1998). The RP-ACS1 protein contains the conserved serine residue in the C-terminus, which is phosphorylated in tomato LE-ACS2 and thought to be involved in the regulation of ACS activity and turnover (Tatsuki and Mori, 2001; Chae et al., 2003; Wang et al., 2004). However, activity of other ACC synthases cannot be completely excluded as the genome sequence of R. palustris is largely unknown.
A study of the expression of RP-ACS1 in petioles of plants grown under non-submerged, but otherwise comparable, conditions indicated that the RP-ACS1 mRNA concentration was at a relatively high level at the beginning of the day and decreased during the subsequent hours (Figs 2C, 4C). So, although the RP-ACS1 transcript level did not increase in the shoot during the first hours of submergence, it was concluded that flooding maintains the messenger concentration at a discrete level and thus makes it possible for the ACC concentration to increase in the plant relative to the control.
Further analysis of the localization of the messenger during submergence showed that the ACC synthase transcript was most abundant in the petioles, the part of the plant that elongates most after submergence (Voesenek et al., 1990a), but that it also started to accumulate in the lamina after 24 h under water (Fig. 3). It has been found previously that ACC oxidase mRNA, and activity upon submergence, are also localized in the petioles (Vriezen et al., 1999), which indicates that ACC synthesis occurs mainly at the site of ethylene production. Although localized ethylene production does not seem to be useful during complete submergence, as ethylene accumulates in the whole plant (Banga et al., 1996), it is probably very useful to ensure high ethylene concentrations in the petioles when the plant is only partly submerged and the ethylene produced can diffuse away freely (Voesenek et al., 2003b). Comparable results were obtained by Zarembinski and Theologis (1997) who found within 12 h of partial submergence an induction of OS-ACS1 mRNA in the cell elongation zone of deep-water rice internodes.
Rhythmic ethylene production and ethylene biosynthesis gene expression
In non-submerged, soil-grown R. palustris plants ethylene production showed a clear rhythm with the highest levels in the dark and the lowest level under light conditions (Fig. 4E). In many plants ACC synthase activity has been shown to be rate-limiting in ethylene production under non-stressed conditions (Yang and Hoffman, 1984; Kende, 1993), so the observation that ethylene production is correlated with RP-ACS1 messenger levels in R. palustris plants suggests that ACC activity is regulated at the transcript level under these conditions. By contrast, under submerged and hypoxic conditions, or in cases of high ethylene production, ACC oxidase activity has been found to be rate-limiting (English et al., 1995; Yamamoto et al., 1995; Barry et al., 1996; Lasserre et al., 1996; Vriezen et al., 1999). Interestingly, waterlogged or hydroponnically grown R. palustris plants produce more ethylene, with a rhythm that is opposite to that found in soil-grown plants (Voesenek et al., 1990a, 1997), and closely resembles the fluctuations in RP-ACO1 messenger levels as determined in this study. The function of rhythmic ethylene production in plants remains unclear. During submerged conditions, the accumulated ethylene induces growth of the petioles (Voesenek et al., 1990b). The low amount of ethylene produced in R. palustris under drained conditions may also have a slight growth-stimulating effect, as petiole growth during the dark phase seems to be slightly higher than during the light phase (Voesenek et al., 1997). Moreover, the mRNA level of a growth-associated R. palustris -expansin gene, Rp-EXP1, also shows a circadian pattern comparable to ethylene release. This gene is known to be responsive to ethylene (Vriezen et al., 2000), suggesting that the plant responds to the rhythm of the basal ethylene production.
The messenger accumulation pattern of the ethylene biosynthetic genes showed that they were regulated by multiple signals. The (initial) persistence of the rhythms in RP-ACS1 and RP-ACO1 transcript levels under constant light (Fig. 4B), indicates an influence of the circadian clock on the mRNA levels. Unlike the CAB gene, however, the amplitude of the RP-ACS1 and RP-ACO1 rhythm decreased strongly when the dark phases were omitted. This means that, next to the circadian clock, a diurnal signal also influenced the messenger levels. The pathway that controls rhythmic expression of the ethylene biosynthetic genes in R. palustris and other species is still unknown, but may act via a sub-group of MYB-related transcription factors that were shown to be involved in the regulation of circadian gene expression in Arabidopsis (Schaffer et al., 1998; Wang and Tobin, 1998; Kuno et al., 2003).
Conclusion
The regulation of ethylene biosynthesis in R. palustris and other plants is complex because many genes encoding for ACC synthase and ACC oxidase and many other factors may influence ethylene release. In this report, the rhythmic patterns of ethylene synthesis, and of RP-ACS1 and RP-ACO1 mRNA accumulation, are shown in soil-grown, non-submerged R. palustris. Under these growth conditions, ACC synthesis is expected to be rate-limiting, and the pattern of RP-ACS1 mRNA accumulation correlated with ethylene production. Submergence, which results in rapid changes of many physiologically important parameters, like the level of oxygen and carbon dioxide, induces ACC synthase activity in the short term, possibly via post-transcriptional or post-translational regulation. Increased ACC synthase activity, together with a decreased ACC oxidase activity, causes accumulation of ACC in the shoot during submergence.
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Acknowledgements |
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Part of this work was supported by the Dutch Science Foundation (Pionier grant no. 800.84.470 to LACJV). We wish to thank Kees Blom for critically reading this manuscript.
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Footnotes |
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* Present address: Rothamsted Research, Division Crop Performance and Improvement, Harpenden, Herts AL5 2JQ, UK.
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References |
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