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First published online June 9, 2006; 10.1104/pp.106.082636 Plant Physiology 141:1644-1652 (2006) © 2006 American Society of Plant Biologists Long-Term Submergence-Induced Elongation in Rumex palustris Requires Abscisic Acid-Dependent Biosynthesis of Gibberellin11Plant Ecophysiology, Institute of Environmental Biology, Utrecht University, Utrecht, Sorbonnelaan 16, 3584 CA, The Netherlands (J.J.B., J.B., A.J.M.P., N.W., K.G., L.A.C.J.V.); Rothamsted Research, Harpenden, AL5 2JQ Herts, United Kingdom (D.W., P.H.); and Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, S–90183 Umea, Sweden (T.M.)
Rumex palustris (polygonceae) responds to complete submergence with enhanced elongation of its youngest petioles. This process requires the presence of gibberellin (GA) and is associated with an increase in the concentration of GA1 in elongating petioles. We have examined how GA biosynthesis was regulated in submerged plants. Therefore, cDNAs encoding GA-biosynthetic enzymes GA 20-oxidase and GA 3-oxidase, and the GA-deactivating enzyme GA 2-oxidase were cloned from R. palustris and the kinetics of transcription of the corresponding genes was determined during a 24 h submergence period. The submergence-induced elongation response could be separated into several phases: (1) during the first phase of 4 h, petiole elongation was insensitive to GA; (2) from 4 to 6 h onward growth was limited by GA; and (3) from 15 h onward underwater elongation was dependent, but not limited by GA. Submergence induced an increase of GA1 concentration, as well as enhanced transcript levels of RpGA3ox1. Exogenous abscisic acid repressed the transcript levels of RpGA20ox1 and RpGA3ox1 and thus inhibited the submergence-induced increase in GA1. Abscisic acid had no effect on the tissue responsiveness to GA.
In several semiaquatic species ethylene promotes shoot extension to rates many times faster than normal (Musgrave et al., 1972  ; Kende et al., 1998; Voesenek et al., 2003a). In these plants, ethylene accumulates to high levels inside the submerged tissue and acts as a signal for submergence (Voesenek et al., 1993). The enhanced elongation response in these species functions as part of an underwater escape mechanism that enables submerged shoots to emerge from the water and function as snorkels, thus restoring gas exchange between the atmosphere and the submerged plant tissues (Voesenek et al., 2004).
The dicot Rumex palustris also responds to submergence with this strong enhancement in cell elongation of petioles (Vreeburg et al., 2005
The biosynthetic pathway for GA1, which is the major bioactive GA in Rumex (Rijnders et al., 1997
Regulation of GA levels takes place predominantly through the activities of GA20ox, GA3ox, and GA2ox. Overexpression of genes encoding earlier enzymes in the biosynthetic pathway generally does not affect plant stature: Feedforward and feedback regulation of GA20ox and GA2ox transcript levels enables the plant to maintain stable GA levels despite large changes in earlier metabolites (Fleet et al., 2003 In this project we investigated whether submergence and ABA regulate GA biosynthesis in petioles of R. palustris. For this work, orthologs for GA20ox, GA3ox, and GA2ox were cloned from this species. Subsequently, the kinetics of transcription of these genes was measured in detail during 24 h of submergence and was compared to changes in concentration of bioactive and inactive GAs and to a detailed analysis of elongation growth. In addition, the effect of submergence and ABA on GA sensitivity was investigated. The results show that the underwater elongation response could be separated in distinct GA-insensitive and GA-dependent phases. Submergence induced an accumulation of GA1 and enhanced transcript levels of RpGA3ox1. These increases in GA1 content and RpGA3ox1 transcript levels were inhibited when submerged plants were treated with ABA, whereas ABA did not affect GA sensitivity.
Submergence-Induced Elongation Upon complete submergence of R. palustris, leaf elongation rates increased strongly, after a lag phase of 2 h (Fig. 1 ), to a rate almost 4 times that of control plants (after 4 h of submergence). After this point, the growth rate decreased slightly, but increased again after 8 h, after which the elongation rate remained 2- to 3-fold faster than air-grown plants for at least 22 h. Separate measurements showed that after 2 d of submergence, petiole length increased almost 3-fold (Fig. 1 insert), whereas leaf blades grew only by 30%.
Analysis of GA Concentrations To examine whether the enhanced elongation in petioles correlated with enhanced GA biosynthesis, the levels of bioactive GA1 as well as its precursors (GA53, GA19, and GA20) and first catabolite (GA8) were measured. The level of GA53 was increased during submergence after a lag phase of 2 h (Fig. 2A ). Changes in GA19 and GA20 concentration followed a similar pattern: an increase in GA19 was observed after 4 to 6 h, followed by an increase in GA20 after 8 h (Fig. 2, B and C). The concentration of bioactive GA1 increased 2-fold in submerged petiole tissue after only 4 h (Fig. 2D). After this point the concentration remained above that in air-grown plants during the remainder of the treatment period. GA8, the first catabolite of GA1, was present at a relatively high concentration (Fig. 2E). Apart from a transient increase in submerged plants between 4 and 8 h of treatment, GA8 decreased gradually in both submerged and air-grown tissue during the treatment period.
Regulation of GA Biosynthesis during Submergence
To determine if the submergence-induced changes in GA concentration resulted from enhanced transcript levels of GA-biosynthetic genes, orthologs from GA20ox, GA3ox, and GA2ox were cloned from R. palustris and their mRNA levels were analyzed by means of real-time reverse transcription (RT)-PCR in submerged and air-grown plants. To isolate cDNA clones, PCR was performed on a cDNA library from R. palustris shoots submerged for 24 h, using degenerated oligonucleotides based on conserved domains of orthologs from Arabidopsis and potato (Solanum tuberosum). Full-length cDNAs were obtained by screening the cDNA library and extension by PCR. We named these genes RpGA20ox1, RpGA3ox1, and RpGA2ox1, respectively. Their predicted amino acid sequences contained the residues necessary for the binding of Fe2+ and the cofactor 2-oxoglutarate conserved in all 2-oxoacid-dependent dioxygenase proteins (Prescott and John, 1996 Real-time RT-PCR analysis of RpGA20ox1, RpGA3ox1, and RpGA2ox1 showed that submergence induced distinct alterations of transcript levels of the three genes. Transcript levels of RpGA20ox1, whose product catalyzes the consecutive conversions from GA53 via GA44 and GA19 to GA20, decreased upon submergence (Fig. 3A ). After 4 h of submergence RpGA20ox1 transcript levels increased again gradually to levels equal to or above those found in air-grown plants. Transcript levels of RpGA3ox1, whose product catalyzes the conversion of GA20 to GA1, increased rapidly inside the submerged tissue. After a lag phase of about 1 h, RpGA3ox1 transcript abundance peaked to a level 4-fold higher than that of air-grown plants (Fig. 3B). A partial fragment of a second RpGA3ox clone demonstrated no change in transcript levels upon submergence (data not shown). Transcript levels of RpGA2ox1, whose product inactivates GA1 by conversion to GA8, also increased in submerged plants (Fig. 3C). However, after 4 to 6 h of submergence, RpGA2ox1 transcript levels decreased to levels below those in air-grown plants.
Effect of ABA on GA Biosynthesis and Catabolism
Externally applied ABA has been shown to inhibit the increase in GA1 concentration in submerged plants (Benschop et al., 2005
Effect of ABA on GA Sensitivity
To investigate whether ABA influenced GA sensitivity as well as GA biosynthesis, plants were submerged in solutions containing a matrix of concentrations of GA and ABA, an approach similar to that described for deepwater rice (Hoffmann-Benning and Kende, 1992
Manipulation of in Vivo GA and ABA Concentrations
Submergence induced an enhancement of petiole elongation after a lag time of 2 h (Fig. 1), but the increase in GA1 concentration could not be observed until at least 4 h of submergence (Fig. 2). Therefore, the increase in growth rate during the initial hours of submergence could not be explained by increased GA1. Nevertheless, the enhanced growth that took place immediately following submergence might still be mediated by GA because sensitivity to GA was shown previously to increase upon submergence (Rijnders et al., 1997 Underwater elongation was not affected by GA treatment during the initial growth phase (2–4 h; Fig. 7A ). However, the temporary slowing of growth rate that was observed in untreated plants during the period of 4 to 10 h of submergence was partly prevented by externally applied GA (Fig. 7A). After 12 h of submergence the growth rate of GA-treated plants was only slightly higher than or even equal to the rate found in untreated plants.
Pretreating plants with paclobutrazol showed that the initial growth phase (2–6 h after submergence) was almost unaffected by this treatment (Fig. 7B). Only a very high dose (100 µM) of paclobutrazol was able to reduce growth somewhat during this period. However, this concentration was likely to have affected processes other than GA biosynthesis, as the growth rate of these leaves was also inhibited before plants were submerged (see Fig. 7B; t < 0), and separate experiments showed that the inhibitory effect on growth by this high concentration of paclobutrazol could not be fully rescued by external GA in these plants (data not shown). During the second period of fast elongation (6–18 h) growth was significantly inhibited even at low concentrations of paclobutrazol. Although some underwater elongation still remained, the growth rates were only marginally faster than those measured just before the start of submergence (Fig. 7B). Taken together, these results suggest that the enhanced elongation response consist of an initial, GA-insensitive phase (0–4 h), a second phase of GA-limited growth (4–15 h), and a third phase that is also dependent, but not limited by GA signaling (from 15 h onward). Although GA did not influence enhanced elongation during the first few hours after submergence, growth in this period was inhibited by ABA (Fig. 7C). Application of 3 µM ABA to submerged plants reduced the enhanced elongation response by more than 50%.
We present here a detailed analysis of regulation of GA1 biosynthesis in petioles of R. palustris during the first 24 h of submergence. The submergence treatment induced changes in transcript levels of a number of different GA biosynthesis genes, and these were reflected in the changes of endogenous GA levels, ultimately leading to a 2-fold increase in bioactive GA1. This increase in GA1 was consistent with the onset of a GA-dependent growth phase after 4 h, and was dependent on ethylene-induced down-regulation of ABA levels in submerged petioles.
We observed a sharp increase in RpGA3ox1 transcript levels in petioles within 2 h of submergence, indicating an enhanced capacity for GA1 biosynthesis (Fig. 3). Consistent with this observation, the concentration of GA1 increased after 4 h of submergence (Fig. 2). The increase in RpGA3ox1 transcript levels following submergence was accompanied by a decrease in transcript levels of RpGA20ox1 and an increase in that of RpGA2ox1 (Fig. 3). These patterns in transcription upon submergence were confirmed in an independent experiment (Fig. 5).
The reduced RpGA20ox1 transcript levels may explain the accumulation of GA53 (Fig. 2), but there is no corresponding decrease in the concentrations of GA20 and GA19, which suggested that there may be enhanced activity (concentration and/or flux) at upstream steps in the pathway earlier than GA53. The changes in transcript levels of RpGA20ox1 and RpGA2ox1, which are opposite to what would be expected to increase GA biosynthesis, may be the consequence of negative feedback and positive feedforward regulation, respectively, in response to increased GA signaling, which allows GA homeostasis (for review, see Hedden and Phillips, 2000
Alternatively, it could be argued that more orthologs of each gene might be present in the Rumex genome, which might be regulated differently from the ones presented here. Although it is impossible to fully exclude this possibility, it should be noted that the genes presented here for R. palustris were the sole members that could be identified from a screen on cDNA from submerged petiole tissue and genomic DNA. The lack of additional members is in agreement with the notion that GA biosynthesis and breakdown genes are generally members of small gene families in species completely sequenced so far (Sponsel and Hedden, 2004
In paclobutrazol-pretreated plants no significant interaction between ABA and GA was observed (Fig. 6). This suggests that externally applied ABA is not affecting the responsiveness of R. palustris petioles to GA. This contrasts with work in the submergence-tolerant deepwater rice, in which it was suggested that ABA inhibits sensitivity to GA (Hoffmann-Benning and Kende, 1992
Instead of reducing GA sensitivity, ABA inhibited transcription of RpGA20ox and RpGA3ox (Fig. 5). As a result of this, the increase in GA1 that takes place in submerged petioles is absent in ABA-treated plants (Fig. 4). This increase in GA1 is crucial for enhanced underwater elongation as long-term extension growth depends strongly on GA (Fig. 7B). In previous work we showed that ABA declines rapidly in petioles of R. palustris upon submergence or ethylene treatment (Benschop et al., 2005
The observation that submergence-induced elongation precedes an up-regulation of GA1 (Fig. 1 versus Fig. 2) indicated that elongation growth during this phase was not dependent on this increase in GA. This was confirmed by the observation that paclobutrazol and external GA could not alter elongation growth during this initial period (Fig. 7, A and B). In contrast, elongation growth during this period was inhibited by ABA (Fig. 7C). The pharmacological treatments showed that submergence-induced elongation in R. palustris can be separated into several phases. The first phase starts 2 h after submergence and is characterized by a rapid increase in elongation rate. This initial burst in growth is insensitive to GA as it remains unaffected by manipulations of GA concentration. Although our paclobutrazol-pretreated plants showed a very severe GA-deficient phenotype we can, however, not completely exclude that trace amounts of GA1 still present play a role in petiole elongation during the first hours of submergence. The second growth phase is between 5 and 15 h of submergence. Here elongation is limited by GA and can be directly manipulated by changing the GA content (Fig. 7, A and B). A third phase, from 15 h onward, is dependent on, but not limited by GA signaling. In this phase the GA concentration and GA sensitivity seem to be saturated, since adding extra GA does not further enhance elongation. Removing GA during this phase does, however, inhibit elongation severely, indicating a continued dependency on GA.
We examined the role of GA during submergence-induced elongation in R. palustris at a molecular, biochemical, and physiological level. Submergence induced an increase in GA1 content in petioles of R. palustris probably through enhanced transcription of RpGA3ox1. This up-regulation is mediated by ABA, which acts as an inhibitor of RpGA3ox1 transcription. High levels of ethylene, that accumulate upon submergence, induce a rapid decline in ABA (Benschop et al., 2005 ). This process relieves the inhibitory effect on RpGA3ox1 and allows the concentration of GA1 to increase, which is a requirement for long-term elongation in submerged Rumex plants.
Plant Growth and Elongation Measurements
Rumex palustris plants were grown according to Benschop et al. (2005)
For measurements of leaf elongation rates, linear displacement transducers were used according to Voesenek et al. (2003b
Levels of GAs in petiole and leaf samples were analyzed using 10 mg dry weight of previously ground and freeze-dried tissue. [2H2]GAs were added to the samples as internal standards, in amounts of 375 pg for GA12, GA15, GA24, GA9, GA4, and GA34 and 750 pg for GA53, GA44, GA19, GA20, GA29, GA1, and GA8. GAs were extracted in 80% methanol with 0.02% diethyl dithiocarbamate as antioxidant with vigorous shaking for 1 h at 4°C. The mixture was centrifuged at 2,800 rpm for 10 min at 4°C. The supernatant was transferred into kimble tubes and dried under reduced pressure. The residue was redissolved in 50 µL methanol; 300 µL hexane was added and the mixture was applied to a Silica (1 g) column previously conditioned with hexane and equilibrated with hexane/ethyl acetate (80:20). GAs were eluted with methanol containing 1% acetic acid. The eluate was dried under reduced pressure and methylated with ethereal diazomethane. Dried residues were redissolved in 200 µL 30% methanol containing 1% acetic acid. When necessary samples were filter centrifuged through an Ultrafree-MC centrifugal filter device (0.22 µm; Millipore) before separation on a C18-reverse HPLC column, in a gradient of 30% methanol to 100% methanol. Fractions were grouped, dried under reduced pressure, and transferred to gas chromatography vials. Samples were trimethylsilylated and GA analysis was carried out by gas chromatography/mass spectrometry selected reaction monitoring using a JEOL/SX/102A four-sector mass spectrometer (JEOL) according to Moritz and Olsen (1995)
The isolation of cDNAs encoding GA-biosynthetic enzymes was carried out by PCR amplification using degenerate primers based on the most conserved regions of known orthologs of Arabidopsis (Arabidopsis thaliana). For GA20ox primers 5'-GAGAAGCTTACNGGNCCNCAYWSNGAYCC-3' and 5'-GCGAATTCNCCDATRTTNACNACYAA-3' (from the conserved amino acid sequences TGPHCDP and LVVNIG) were used. For GA3ox primers 5'-GCSATGGGSCTSGCSGCSCAYACNG-3' and 5'-CCGTTHGASAGRTGGAASAGRTCNCC-3' (corresponding to MGLAAHTD and GDLFHIL) were used, and for GA2ox primers 5'-AAYGGNGAYNTNGGNTGG-3' and 5'-CTRAANCCRTTNGTCATNAC-3' (corresponding to NGDIGW and FGEHTDP) were used. PCR products amplified from genomic DNA or cDNA from R. palustris shoots were cloned in a pGEM-T easy vector (Promega) and sequenced. Positive clones were labeled as probes to screen the cDNA library and the isolated partial cDNAs were extended to full length by nested PCR using specific primers from the gene and from the vector.
For each sample, RNA of five 3rd petioles was extracted using a modified method of Kiefer et al. (2000)
Preparation of Lysate
Enzyme Assays
GA3 (Duchefa), (±)-ABA (Sigma), and paclobutrazol (Duchefa) were dissolved in ethanol or acetone to a stock concentration of 25 mM (for ABA) or 100 mM (for GA and paclobutrazol) and diluted at least 1,000-fold with distilled water. Paclobutrazol pretreatment occurred once, 96 h before measurements, with 10 mL 100 µM paclobutrazol or (for controls) with 0.1% (v/v) ethanol. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: RpGA20ox1 (DQ641498); RpGA3ox1 (DQ641497); and RpGA2ox1 (DQ641499).
We thank Rob Welschen for technical assistance and Ronald Pierik and Frank Millenaar for discussions and comments. Received April 27, 2006; returned for revision May 22, 2006; accepted May 31, 2006.
1 This work was supported by the Dutch Science Foundation (PIONIER grant no. 800.84.470) and by a grant from the European Union (HPRM–CT–RTN1–2000–00090). 
2 These authors contributed equally to the paper.
3 Present address: Department for Physiological Chemistry, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.
4 Present address: Laboratori de Genètica Molecular Vegetal, Consorci Consejo Superior de Investigaciones Científicas-Institut de Recerca i Technologia Agroalimentàries, Jordi Girona, 18–26, 08034 Barcelona, Spain.
5 Present address: Institute for Water and Wetland Research, Faculty of Science, Radboud University, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Laurentius A.C.J. Voesenek (l.a.c.j.voesenek{at}bio.uu.nl). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.082636. * Corresponding author; e-mail l.a.c.j.voesenek{at}bio.uu.nl; fax 31–30–2518366.
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ABSTRACT
RESULTS
-galactoside (final concentration 1 mM) and the cultures grown for a further 3 h at 25°C. The cells were then pelleted by centrifugation at 5,000 rpm at 4°C for 5 min, the pellets frozen and thawed, and then incubated in 1 mL of freshly prepared lysis buffer containing 100 mM Tris at pH 7.5, 5 mM dithiothreitol, and 1 mg mL–1 lysozyme, for 15 min at room temperature. The lysate was then incubated for a further 15 min after the addition of 20 units DNase. The lysate was then centrifuged at 15,000g for 10 min and the supernatant transferred to a separate tube. 
