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Plant Physiol, February 2001, Vol. 125, pp. 955-968 A Comparative Molecular-Physiological Study of Submergence Response in Lowland and Deepwater Rice1Vakgroep Moleculaire Genetica and Departement Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium (D.V.D.S., Z.Z., M.C.V.M.); and Laboratorium voor Plantenfysiologie, Universitaire Instelling Antwerpen, Universiteitsplein 1, B-2610 Wilrijk, Belgium (E.P., H.A.V.O.)
Survival of rice (Oryza sativa) upon an extreme rise of the water level depends on rapid stem elongation, which is mediated by ethylene. A genomic clone (OS-ACS5) encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, which catalyzes a regulatory step in ethylene biosynthesis, has been isolated from cv IR36, a lowland rice variety. Expression was induced upon short- and long-term submergence in cv IR36 and in cv Plai Ngam, a Thai deepwater rice variety. Under hypoxic conditions, abscisic acid and gibberellin had a reciprocal opposite effect on the activity of OS-ACS5. Gibberellin up-regulated and abscisic acid down-regulated OS-ACS5 mRNA accumulation. Growth experiments indicated that lowland rice responded to submergence with a burst of growth early on, but lacked the ability to sustain elongation growth. Sustained growth, characteristic for deepwater rice, was correlated with a prolonged induction of OS-ACS5. In addition, a more pronounced capacity to convert ACC to ethylene, a limited ACC conjugation, and a high level of endogenous gibberellin20 were characteristic for the deepwater variety. An elevated level of OS-ACS5 messenger was found in cv IR36 plants treated with exogenous ACC. This observation was concomitant with an increase in the capacity of converting ACC to ethylene and in elongation growth, and resulted in prolonged survival. In conclusion, OS-ACS5 is involved in the rapid elongation growth of deepwater rice by contributing to the initial and long-term increase in ethylene levels. Our data also suggest that ACC limits survival of submerged lowland rice seedlings.
Rice (Oryza sativa) is a
semiaquatic plant adapted to survive submergence for a certain period
of time. Rice is classified into three ecotypes according to its water
requirement and tolerance: highland, lowland (irrigated or rain-fed),
and deepwater rice (Takahashi, 1984 Rapid elongation in response to submergence is triggered by
oxygen deficiency and is controlled by ethylene (Kende et al., 1998 The studies mentioned above were focused on the growth of internodes of
rice plants aged between 3 and 13 weeks. As a consequence, very little
is known on the submergence response of seedlings. Yet in those cases
where seedlings live under fully submerged conditions for several days,
yield losses are considerable. Submergence is indeed a major constraint
to rice production (Widawsky and O'Toole, 1990 High-nutritional and high-yielding lowland cultivars could be adapted by genetic engineering, allowing them to grow in flooded areas or tidal swamps. As a first step toward this goal we have studied an ACS gene (OS-ACS5) that is induced by submergence in lowland and in deepwater rice. By correlating growth with expression patterns for submergence-induced ACS, in vitro ACC oxidation capacity, and levels of ACC, ethylene, ABA, and GA, we have tried to underpin the molecular and biochemical alterations that contribute to the difference in flooding tolerance. Although not being limited to the response of seedlings, our study pays particular attention to the reaction in early development.
Isolation and Characterization of OS-ACS5, a Genomic ACS Clone from Lowland Rice (cv IR36) A genomic library of cv IR36 was screened at low stringency with
ACS cDNAs from deepwater rice (cv Plai Ngam; Van Der Straeten et al.,
1997 A comparison of the OS-ACS5 promoter with that of
LE-ACS3, which is responsive to submergence in tomato (Olson
et al., 1995
Genomic DNA Blot of OS-ACS5 for Lowland and Deepwater Rice To reveal the existence of a closely related member in the cv IR36 ACS gene family, a genomic DNA gel blot was performed under very stringent conditions. Genomic DNA from cv IR36 and cv Plai Ngam was digested with EcoRI, BglII, XhoI, and EcoRI/PstI. Single bands hybridized upon digestion with XhoI, EcoRI/PstI, and EcoRI, with sizes corresponding to those predicted from the sequence (data not shown). The digestion with BglII yielded two predicted bands of 4.3 and 2.9 kb. The intensity of the bands correlated to a single-copy gene according to a copy number reconstruction analysis. Deepwater rice had a pattern identical to that of cv IR36 in all lanes, supporting the existence of an OS-ACS5 ortholog in these plants. Developmental and Stress-Regulated Expression of OS-ACS5 Figure 2A shows the expression
of OS-ACS5 at different stages of vegetative growth in cv
IR36. A significant stimulation was found when plants at any age were
submerged for 4 h. However, a peak in mRNA accumulation occurred
in 5-week-old submerged plants. To determine whether OS-ACS5
plays a role in the initial growth-promoting increase in ethylene
synthesis, short-term submergence induction was investigated and
compared with OS-ACS1, which is induced within 12 h of
submergence and by low oxygen treatment (Zarembinski and Theologis,
1997
When plants were treated with 10 µM cycloheximide for
4 h followed by 6 h of incubation without the translation
inhibitor, a 3-fold induction of OS-ACS5 mRNA was observed
(data not shown). An increase of the steady-state mRNA level upon
cycloheximide treatment was also demonstrated for the
OS-ACS1, OS-ACS2, and OS-ACS3 genes
(Zarembinski and Theologis, 1993 Comparison of the OS-ACS5 mRNA Level and Growth in cv IR36 and cv Plai Ngam Figure 3 presents the biometric growth data of 9-d-old (Fig. 3B) and 9-week-old (Fig. 3A) cv Plai Ngam or cv IR36 exposed to plant hormones (GA, ABA, and ethylene) in air or in a low-oxygen atmosphere for 2 d. Hypoxic treatment (mimicking the effect of submergence), as well as exposure to GA and ethylene all stimulated growth. A growth increase of 2- to 3-fold was observed upon exposure to GA in air. In contrast, supplementing ABA suppressed the growth by a factor of 2 to 4 (with the exception of 9-d-old cv IR36, where growth remained at control level). This suppression was not released by combination with a hypoxic treatment.
The steady-state mRNA level of OS-ACS5 was assessed from the same samples (Fig. 3). Up- and down-regulation of expression was correlated with a positive or negative effect on growth, although this was less pronounced in hypoxia-treated seedlings. In 9-week-old cv Plai Ngam plants subjected to a hypoxic atmosphere, OS-ACS5 mRNA was 5-fold induced. In air, GA and ethylene increased the steady-state level of transcription 2- to 3-fold, whereas in hypoxia, an 8-fold stimulation was noticed. ABA reduced the amount of mRNA to roughly 20% of the control level in air and low oxygen (Fig. 3A, top). This effect of ABA was also observed in 9-week-old cv IR36 plants (Fig. 3A, bottom), albeit only at subambient oxygen concentration. The influence of GA was similar to that found in cv Plai Ngam. However, the combined treatment with hypoxia and ethylene did not result in an equal stimulation of the mRNA level when compared with cv Plai Ngam. The difference in growth response to ABA between these two cultivars was more obvious in young rice seedlings (Fig. 3B). In cv Plai Ngam, addition of ABA suppressed the elongation in air by 70%. This reduction was even more severe in low oxygen (about 8-fold). In contrast, cv IR36 seedlings were more tolerant to ABA. No significant reduction in growth could be observed in air and only 40% in hypoxia. Nevertheless, a suppression of OS-ACS5 mRNA upon ABA treatment was noticed in cv Plai Ngam and in cv IR36. Deepwater and Lowland Rice Seedlings Respond Differently to Sustained Submergence To mimic the natural growth situation in deepwater areas, a sustained submergence of 2 weeks was conducted on both cultivars at the age of 9 d. Figure 4 shows a record of the daily increase in height during the first 10 d of submergence. When grown in air, the elongation rate increased gradually, reaching a peak at d 4 or 5 in cv IR36 or in cv Plai Ngam, respectively. However, when submerged, cv IR36 had the most pronounced elongation on d 1. After 3 d, growth was strongly reduced and almost ceased after d 5 (Fig. 4, bottom). In contrast, the growth rate of cv Plai Ngam increased until d 3. A significant growth was observed all along the recording period (Fig. 4, top). The two varieties showed a different phenotypic appearance during the course of the experiment. The leaves of cv IR36 were already turning yellowish after 2 d of submergence and the plants were entirely chlorotic within a week. In contrast, cv Plai Ngam kept growing even after 10 d of submergence, although leaves turned light green or yellowish around d 7.
Exogenous ACC Treatment Results in a Constant Up-Regulation of OS-ACS5 in Lowland and Deepwater Rice upon Long-Term Submergence and Correlates with an Increased Elongation Growth Figure 5 (A and B, insets) shows RNA gel blots of tissues submerged for 2, 7, and 14 d in the absence or presence of 20 µM ACC, probed with OS-ACS5 and OS-ACS1. In cv Plai Ngam and in cv IR36, a sustained high level of OS-ACS5 mRNA was observed when ACC was added. In the lowland variety the average induction was approximately 3-fold; in the deepwater cultivar it was at least 6-fold. In both cases the enhanced accumulation of OS-ACS5 was correlated with an increase in length. Ten days after submergence in the presence of ACC, the average additional increase in height was 33% for cv IR36 and 23% for cv Plai Ngam, compared with the values without exogenous ACC.
Furthermore, in the absence of ACC, OS-ACS1 was significantly induced between 2 and 7 d in cv Plai Ngam, but not in cv IR36. However, it should be noted that the actual accumulation was again approximately an order of magnitude below that for OS-ACS5 (exposure for OS-ACS1 was 10-fold longer than for OS-ACS5). In contrast, upon addition of ACC, OS-ACS1 mRNA levels remained at control level in cv Plai Ngam over the entire period of submergence, whereas in cv IR36 an induction was seen after 14 d. Exogenous ACC Treatment Extends the Life Span of Submerged Lowland Rice When seedlings were subjected to exogenous ACC, a delay in chlorosis was remarked in cv IR36. Therefore, chlorophyll levels were measured in both varieties after 2, 7, and 14 d of submergence in the presence or absence of 20 µM ACC. Figure 6 shows the chlorophyll levels relative to the level prior to submergence. No statistically significant differences were observed for cv Plai Ngam. Final levels remained close to 50% of the initial value. In contrast, cv IR36 plants displayed a clear retention of chlorophyll. After 14 d of submergence, the level had dropped to only 30%, against 14% in non-treated controls. As a consequence, plants looked healthier when 20 µM of ACC was supplied.
Submergence-Induced ACC Oxidase (ACO) Activity and Ethylene Emanation in Rice Seedlings To assess the capacity of rice plants to convert the ethylene
precursor ACC, in vitro ACO activity was measured (Vriezen et al.,
1999
Likewise, ethylene emanation most significantly increased in seedlings after 2 d of submergence (Fig. 7, C and D). Inductions were more elevated for cv Plai Ngam (3-fold), whereas a 2-fold increase was observed in cv IR36. When 20 µM ACC was administered during submergence, a 40% to 50% increase in ethylene release was found in the two cultivars. ACC and Conjugated ACC Contents in Air-Grown and Submerged Lowland and Deepwater Rice To verify the physiological relevance of the expression of the
OS-ACS5 gene in submerged rice, the levels of free ACC and conjugated ACC were measured (Martin et al., 1995
In addition, a significant difference was observed in the levels of conjugated ACC at each time point of submergence. Formation of ACC conjugates was always higher in cv IR36 than in cv Plai Ngam. Even after 2 weeks of submergence, the levels of conjugated ACC were still 65% higher in cv IR36. ABA and GA Contents in Air-Grown and Submerged Lowland and Deepwater Rice The concentration of endogenous ABA and GA and the balance
between these hormones are determining factors in
submergence-stimulated elongation growth of adult rice
(Hoffmann-Benning and Kende, 1992
In view of transferring the "floating trait" from low-yielding
deepwater rice varieties to the high-yielding lowland cultivars, a
comparison of the molecular aspects of ethylene biosynthesis in both
types would be valuable. Here we have correlated the expression of two
submergence-responsive ACS genes with growth patterns and with in vitro ACC oxidation, levels of ACC, ethylene, ABA, and GA. All
traits were monitored in lowland and in deepwater rice seedlings. A
limited set of experiments was also conducted on growing stem segments
of adult plants. Basic similarities were found in the submergence
response of seedlings and adult tissues at the molecular level,
supporting previous physiological studies (Jackson and Pearce,
1991 In contrast with OS-ACS1, an ACS gene that had
previously been reported to contribute to longer-term ethylene
production upon submergence (Zarembinski and Theologis, 1997 By exposing the plants to subambient oxygen concentrations close to
those found in internodal lacunae of submerged deepwater rice
(Stünzi and Kende, 1989 The effects of ABA and GA on OS-ACS5 gene
expression were generally accompanied by a down- and up-regulation of
growth, respectively (Fig. 3). These data are in accordance with the
view that rapid elongation growth results from an altered balance
between GA and ABA (Hoffmann-Benning and Kende, 1992 Keith et al. (1986) Additional differences between the two cultivars reside at later steps in the pathway (Figs. 7 and 8). The higher level of ACC conjugates in the lowland cultivar could result from the accumulation of ACC, whereas ACO activities are lower than in deepwater rice. A difference in ACC transport may exist, as well. It also remains to be seen whether low oxygen conditions differentially affect the catalytic activity of ACO in the two types. Regulation at the level of ethylene perception can play a role, but the receptors involved have yet to be identified. In conclusion, survival of rice under sustained submergence is modulated at the level of the signaling hormone (ethylene) and the hormone that controls the elongation response (GA). Whether an enhanced ethylene level results in a prolonged survival due to a faster short-term response or as a result of an improved long-term adaptation related to a more optimal maintenance of carbohydrate and energy supplies and a stimulation of aerenchyma formation remains to be investigated.
Plant Material and Growth Conditions Rice (Oryza sativa cv IR36 [lowland rice] and
cv Plai Ngam [deepwater rice]) seeds were soaked in distilled water
and kept at 24°C in the dark for 2 d. Imbibed seeds were
germinated in the dark on vermiculite impregnated with
one-half-strength Hoagland solution (Hoagland and Arnon, 1938 Submergence, Hormone, Wounding, Cycloheximide, and Low-Oxygen Treatments For submergence experiments, 12 rice plants were held upright using a perforated plastic plate in a 2-L glass cylinder filled with one-half-strength Hoagland solution. The solution was refreshed every 2nd d. Plants were grown under the conditions mentioned above. For gene expression analysis, submergence was sustained for 0.5 to 4 h on plants with indicated age (Fig. 2), whereas for comparison of growth data and OS-ACS inductions, as well as the biochemical measurements, 9-d-old seedlings were kept submerged for a period of up to 2 weeks (Figs. 4-8). For RNA gel blots on adult tissues (Fig. 2), entire stems were extracted, with the exception of 13-week-old plants, in which the oldest part (approximately 5 cm) was excluded. Hormonal treatments were on plants of indicated age. Of each growth
regulator (purchased from Sigma, St. Louis), 50 µM were administered by root absorption in one-half-strength Hoagland solution
for 2 d. For mechanical wounding, plants were cut into 1- to 2-mm
pieces with scissors. The cut tissue was kept in a covered Petri dish
on a wet paper (Whatman, Clifton, NJ) for 5 h. The uptake of 10 µM cycloheximide was by root absorption for 4 h,
followed by 6 h in one-half-strength Hoagland solution without the
translation inhibitor. For a comparison of hormonal effects in normal
and in low-oxygen conditions, 20 9-d-old seedlings or 12 stem pieces of
9-week-old plants (16 cm, plus 2 cm of roots) were placed vertically in
a 100-mL beaker filled with 40 mL one-half-strength Hoagland solution
supplemented with 50 µM of the indicated growth regulators. The beaker was placed inside a 4-L glass jar through which
hydrocarbon-free air or a hypoxic atmosphere (6% O2, 3% CO2, and 91% N2) was flushed at a flux rate of
250 mL min Experiments were repeated three to four times. Samples for RNA gel blots were consistently taken at the same time of the day (i.e. 7 h after initiation of the light period) to exclude possible influences of circadian rhythms. Isolation and Sequencing of the OS-ACS5 Gene A cv IR36 genomic library in EMBL-3 Sp6/T7 was used (Clontech,
Palo Alto, CA). Screening was on Hybond N+ (Amersham
Pharmacia Biotech, Little Chalfont, UK) with a radiolabeled equimolar
mixture of three partial cDNA fragments from deepwater rice
ACS genes (Van Der Straeten et al., 1997 PCR Amplification of an OS-ACS5 Promoter Fragment of cv Plai Ngam and a Carboxyl-Terminal Fragment of OS-ACS1 A PCR fragment of the OS-ACS5 promoter of cv Plai Ngam was generated from genomic DNA. Different sets of primers were designed based on the sequence of OS-ACS5 in cv IR36. PCR amplification was performed with a Pyrococcus woesci proofreading DNA polymerase (Roche Diagnostics, Brussels). The reactions (30 cycles each) were performed on a thermocycler OmniGene TR3 CM220 (Hybaid Ltd., Ashford, UK). Genomic DNA from cv IR36 was used as a control. One pair of primers (Z29 and Z35) produced the expected 0.4-kb fragment from cv Plai Ngam. The primers (with melting temperatures) used were 5'-GAGAAAGAGAGAGAGAGTGAGC-3' (55.9°C) and 5'-CAACAGCTTGCCACTCATGATGTTCTC-3' (61.9°C) for Z29 and Z35, respectively. The sequences of two independently amplified DNA fragments were determined with an automated 377 DNA sequencer (Applied Biosystems) using a Bigdye Terminator Sequencing Kit (Applied Biosystems). Using primers Z50 and Z52 on genomic DNA, an OS-ACS1
fragment of 330 bp was amplified from cv Plai Ngam and from cv IR36. Both sequences were identical to the 990- to 1,319-bp fragment of the
OS-ACS1 cDNA (accession no. M96672; Zarembinski and Theologis, 1993 Genomic DNA Gel Blot Genomic DNA was prepared according to Shure et al. (1983) RNA Analysis Total RNA from rice was prepared by the method of Jones et al.
(1985) Ten micrograms of total RNA or 0.5 µg of mRNA was separated on a 1.2% (w/v) agarose gel containing 8% (v/v) formaldehyde. RNA was blotted on Hybond N+ (Amersham Pharmacia Biotech) in 20× SSC. A riboprobe was prepared by T7 RNA polymerase transcription (Riboprobe Gemini II, Promega, Madison, WI). To reach the highest specificity, the 0.5-kb carboxyl-terminal part of the OS-ACS5-coding region was used (2,795-3,364 bp in X97066). This region is highly divergent among the ACS gene family members. A fragment of the same region was used to compare the expression with that of OS-ACS1. Because the average incorporation of radioactivity in probes was above 80% and the length of the OS-ACS1 template was more than one-half that of OS-ACS5 (330 versus 570 bp), it can be concluded that the OS-ACS5 probe had a specific activity (on a molar basis) at most twice as high as that of the OS-ACS1 probe. Therefore, OS-ACS5 signals that were more than 10-fold higher in intensity compared with OS-ACS1 represented an mRNA accumulation of at most an order of magnitude difference. Prehybridization and hybridization were carried out at 45°C in a
buffer containing 50% (v/v) formamide, 3× SSC, 5 × Denhardt's solution, 0.5% (w/v) SDS, and 100 µg mL Determination of Internal Contents of ACC, Conjugated ACC, ABA, GA, and Indole-3-Acetic Acid (IAA) Twenty 9-d-old seedlings were submerged for 4 h, 2 d, 1 week, and 2 weeks. The aerial part was frozen in liquid nitrogen and ground to a fine powder. Approximately 0.5 g of fresh weight was used for each sample. Air-grown plants of the same age were used as a control. Experiments were repeated twice, each with duplicates. ABA, ACC, GA, and IAA were analyzed by a combined solid-phase
extraction procedure based on Prinsen et al. (1991) Determination of ACO Activity and Ethylene Production In vitro ACO measurements were essentially performed according
to Mekhedov and Kende (1996) To measure ethylene emanation entire seedlings (approximately 0.5 g fresh weight) were quickly blotted dry, weighed, and transferred to a
20-mL glass vial with a rubber cap. During these manipulations (a few
minutes) ethylene trapped during submergence treatment could escape,
allowing assessment of ethylene production. However, it should be noted
that although allowing a qualitative comparison between treatments, the
values measured cannot reflect absolute quantitative differences. A
small piece of wet 3MM (Whatman) paper was added to keep the atmosphere
moist. After 2 h, 1 mL of headspace was withdrawn and injected
into a gas chromatographer Di200 (Delsi, Suresnes, France). A linear
response was found up to 4 h of incubation. The calculation of
ethylene production was based on a standard curve obtained from
injections of a gas mixture containing 100 µL L Chlorophyll Determinations Total chlorophyll (a + b) was
measured according to Porra et al. (1989)
The authors gratefully acknowledge Jan Gielen and Wilson Ardiles for DNA sequencing, Gurdev Khush (International Rice Research Institute, Manila, Philippines) and Somboon Anuntalabhochai (Chiang Mai University, Chiang Mai, Thailand) for providing rice seeds (cv IR36 and cv Plai Ngam, respectively), Fumio Takaiwa (National Institute of Agrobiological Resources, Yatabe Ibaraki, Japan) for the rice 17s rDNA, and Ray Wu (Cornell University, Ithaca, NY) for the rice actin 1 cDNA. The authors thank Wim Vriezen (Ghent University, Belgium) for critical reading of the manuscript, Martine De Cock for lay-out, and Karel Spruyt, Rebecca Verbanck, and Christiane Germonprez for art work.
Received July 12, 2000; returned for revision September 1, 2000; accepted October 29, 2000. 1 This research was supported by the European Union (grant no. International Scientific Cooperation China CI1*-CT93-0082) and by the Geconcerteerde Overlegde Acties (grant no. GOA 96016). D.V.D.S. and H.A.V.O. are Research Directors of the Fund for Scientific Research (Flanders).
* Corresponding author; e-mail dostr{at}gengenp.rug.ac.be; fax 32-9-2645349.
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ABSTRACT
INTRODUCTION
ková et al., 1997
258 and 
2
s
-clones were
subcloned in pBluescript KS II as an
EcoRI/XhoI fragment (Stratagene, La
Jolla, CA). Double-stranded DNA sequencing was performed by the dideoxy
chain termination method (Sanger et al., 1977
98 (PTH-ACC) and 223