Journal of Experimental Botany, Vol. 53, No. 379, pp. 2325-2331,
December 1, 2002
© 2002 Oxford University Press
The novel ethylene-regulated gene OsUsp1 from rice encodes a member of a plant protein family related to prokaryotic universal stress proteins
Received 16 April 2002; Accepted 19 July 2002
Institut für Allgemeine Botanik, Universität Hamburg, Ohnhorststrasse 18, D-22609 Hamburg, Germany
1 To whom correspondence should be addressed. E-mail: msauter{at}botanik.uni-hamburg.de
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Abstract |
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Using subtractive hybridization a submergence-induced gene was identified from deepwater rice, OsUsp1, that encodes a homologue of the bacterial universal stress protein family. Sequence analysis revealed that OsUSP1 is most closely related to the bacterial MJ0577-type of ATP-binding USP proteins which have been suggested to act as a molecular switch. USP protein homologues appear to be ubiquitous in plants and are encoded by gene families, but are absent in animal species. In the youngest internode of deepwater rice plants, OsUsp1 expression was very strongly induced within 1 h of submergence. Elevated transcript levels were observed in dividing cells, in expanding cells and in differentiated tissue indicating that USP1 mediates a general process. Gene induction was shown to be regulated by ethylene with a highly similar expression pattern to that observed with submergence treatment. Based on sequence information and on expression data it is hypothesized that OsUSP1 plays a role in ethylene-mediated stress adaptation in rice.
Key words: Deepwater rice, ethylene, MJ0577, Oryza sativa L., submergence stress, universal stress protein.
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Introduction |
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Deepwater rice plants can be induced to grow very rapidly by partial submergence. In southeast Asia where vast areas are flooded yearly during the monsoon season, deepwater rice is a staple crop. Through rapid elongation of the youngest internode the plants keep part of their foliage above the rising water level thus securing access to atmospheric oxygen which is necessary for survival. Growth of the internode is regulated through a signalling cascade that involves several plant hormones. Within 1 h of submergence ethylene accumulates within the plant. Elevated levels of ethylene induce a reduction in abscisic acid levels and an increase in gibberellic acid levels. Both changes together result in the growth-inducing activity of gibberellin (Kende et al., 1998). Within 4 h of submergence the internode starts to elongate at an accelerated rate.
Ethylene not only controls internodal elongation, it is also responsible for a number of additional adaptations to hypoxia such as the growth of adventitious roots at the nodes of the stem (Lorbiecke and Sauter, 1999) and cell death processes during adventitious root emergence (Mergemann and Sauter, 2000) and during aerenchyma formation (Drew et al., 2000). Similar ethylene responses have also been reported for other plant species (Visser et al., 1996; Voesenek and Blom, 1999). In Arabidopsis, it was further shown that the hypoxic induction of alcohol dehydrogenase is partially dependent on ethylene signalling (Peng et al., 2001) suggesting that metabolic adaptation to hypoxia is also, at least in part, regulated by ethylene. Not only hypoxia, but almost all types of biotic and abiotic stresses, induce ethylene biosynthesis. In some cases, as during submergence, a physiological function of stress ethylene has been described. In other cases its role is not understood (Bleecker and Kende, 2000).
In a differential screening for submergence-induced genes in deepwater rice a gene, OsUsp1, has been identified that is regulated by ethylene and may play a role in the adaptation of rice to submergence stress.
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Materials and methods |
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Plant material
Seeds of Oryza sativa L., indica cultivar Pin Gaew 56, were originally obtained from the International Rice Research Institute, Los Baños, Philippines. Rice plants were grown as described (Sauter, 1997). For growth induction by submergence, plants were partially submerged in a 600 l plastic tank filled with tap water leaving approximately 30 cm of the leaf tips above the water surface. For growth induction by hormone treatment, stem sections containing the youngest growth-responsive internode were excised as described by Raskin and Kende (1984) and treated with 150 µM ethephon, or without added hormone as the control for the times indicated. To inhibit ethylene perception, stem sections were treated with 50 µl l–1 norbornadiene (bicyclo-hepta-2,5,-diene; NBD) or with 150 µM ethephon and 50 µl l–1 NBD.
From submerged plants, RNA was isolated from three diferent zones: the intercalary meristem from 0–5 mm above the second highest node, the elongating zone between 5 mm and 15 mm above the second highest node and the differentiation zone just below the youngest node. From stem sections, RNA was isolated either from a 10 mm region above the second youngest node comprising the meristem and part of the elongation zone or from the meristematic zone as described above. Tissue was harvested, frozen in liquid nitrogen and stored at –70 °C until extraction.
Subtractive hybridization
PCR-based subtractive hybridizations were performed according to (Buchanan-Wollaston and Ainsworth (1997) using cDNAs synthesized from mRNA isolated from the intercalary meristem of non-submerged deepwater rice plants as the driver population. Target cDNA populations were obtained from plants which were partially submerged for 2 h or 6 h. In short, mRNAs of the three time points were reverse transcribed with the ‘Time saver cDNA synthesis Kit’ (Amersham Pharmacia Biotech, Freiburg, Germany). The cDNAs were restricted with AluI and RsaI to obtain fragments of approximately 150–400 bp in length. The cDNA fragments were ligated to driver-specific or target-specific adaptors, respectively, and were size-fractionated in an agarose gel. The cDNA fragments were amplified with adaptor-specific primers, and the resulting cDNA populations were used for the subtractive hybridization essentially as described (Buchanan-Wollaston and Ainsworth, 1997). The subtraction procedure was performed through magnetic removal of biotinylated driver/driver or driver/target hybrid cDNAs using streptavidin-coated paramagnetic beads (Dynal, Hamburg, Germany). Target cDNAs which were not hybridized to complementary sequences in the driver population remained in solution and were subsequently amplified with a target-specific primer. After six rounds of enrichment and three times of target-specific cDNA amplification, the resulting cDNAs were cloned into pBluescript (Stratagene, Amsterdam, Netherlands) and sequenced with the ABI PRISMTM Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Weiterstadt, Germany).
5' RACE
The partial cDNA isolated in the differential screening comprised the C-terminal end of the protein coding region and part of the 3'-untranslated region. Rapid amplification of the 5' cDNA end (5' RACE) was employed to obtain a sequence comprising the full-length open reading frame of OsUSP1. For cDNA synthesis mRNA was isolated from the 1 cm zone at the base of the youngest internode of plants that were submerged for 2 h. The gene-specific reverse primer 5'-GACGATCATGACGGAGCAGTG-3' from nt 544 to nt 564 was used for first strand cDNA synthesis. For PCR amplification a second gene-specific primer 5'-AGGAAGGCCC TCTTGATTGC-3' from nt 490 to nt 510 and the forward abridged anchor primer as provided by the manufacturer were used. The resulting PCR products were reamplified using the reverse primer 5'-CGGCCTCCACGTATCTCACCA-3' from nt 299 to nt 319 and the forward abridged universal amplification primer as provided by the manufacturer. The RACE procedure was performed according to the manufacturer’s instructions (Gibco, Eggenstein, Germany).
Sequence analysis
Protein homologues of OsUSP1 were identified in the available protein and nucleic acid databases with the BLAST algorithm (Altschul et al., 1997). Secondary structure prediction was done with the PHD program (Rost, 1996). Phylogenetic analysis was done using CLUSTAL X and TreeView 1.31 (Page, 1996).
RNA blot analysis
RNA was isolated and hybridizations were carried out as described previously (Sauter, 1997). For Northern blot analysis 15–30 µg of total RNA were used. As a control for equal loading, ribosomal RNA was stained with ethidium bromide. For the detection of OsUsp1 transcripts, the 209 bp 3'-terminal fragment isolated in the differential screening was used as a probe. This probe covers the nucleotide sequence from nt 422 to nt 630 of the full-length OsUsp1 cDNA. All steps for hybridization and washing were performed under highly stringent conditions.
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Results |
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Expression of OsUsp1 is induced by submergence
A differential screening with PCR-generated cDNA libraries was performed in order to identify genes which are induced in the intercalary meristem of deepwater rice plants after 2 h or after 6 h of partial submergence. A total of 14 cDNAs was cloned and sequenced. Seven of the partial cDNAs represented the alcohol dehydrogenase 1 gene (ADH1, GenBank Accession No. X16296), two partial cDNAs represented the pyruvate decarboxylase 2 gene (PDC2, GenBank Accession No. U27350) and two partial cDNAs represented a cytosolic gylceraldehyde-3-phosphate dehydrogenase gene homologue (GAPDH). Two partial cDNAs did not have significant homology to known sequences. One partial cDNA represented a gene, OsUsp1, with distinct sequence homology to the bacterial ATP-binding protein MJ0577 (GenBank Accession No. AAB98568) which, in turn, is related to the bacterial universal stress protein family UspA. Up-regulation by submergence was confirmed for three genes, ADH1, PDC2 and OsUsp1 by Northern blot analysis (Fig. 1; data not shown).
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OsUsp1 expression was induced transiently in the internode of rice plants with a peak in transcript levels between 1 h and 2 h after the onset of the submergence treatment (Fig. 1A, B). Transient gene induction was observed in the meristematic zone, in the elongation zone and in the differentiation zone of the youngest, growth-responsive internode (Fig. 1A). Induction of OsUsp1 expression was further analysed in a short-term time-course in the meristem and in the elongation zone (Fig. 1C). The result indicated that OsUsp1 was induced rapidly by submergence with slightly elevated transcript levels appearing after 40 min. Strong induction was observed after 60 min of submergence treatment (Fig. 1C). After a short period of high gene expression, OsUsp1 transcripts were degraded to near basal levels between 2 h and 6 h of submergence (Fig. 1A). A time-course experiment with samples taken every hour between 0 h and 6 h of submergence indicated that transcripts disappeared between 1 h and 4 h in the intercalary meristem and between 2 h and 3 h in the elongation zone (Fig. 1B). In summary, the data indicate that OsUsp1 expression is under stringent control.
OsUsp1 is related to the prokaryotic family of universal stress proteins
A cDNA sequence comprising a full-length open reading frame of OsUSP1 was obtained from the partial cDNA by 5' RACE (Fig. 2; GenBank Accession No. AF491815) using mRNA isolated from the intercalary meristem of plants submerged for 2 h as the RNA source and two gene-specific reverse primers for reverse transcription and for PCR amplification, respectively. The nucleotide sequence obtained was identical except for two nucleotides to a genomic sequence from rice located on chromosome 7 (accession number AP004300). The genomic sequence has two introns at the positions indicated in Fig. 2. Intron 1 has a length of 171 nucleotides and intron 2 has a length of 114 nucleotides.
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Homology searches in EST, cDNA and genomic databases revealed that OsUSP1-like proteins are present in monocot and dicot plants (Fig. 3A; data not shown). The data available further indicated that they are encoded by gene families with at least ten members in rice. A database search was performed on the Arabidopsis thaliana genome. It indicated that Arabidopsis has 17 Usp genes located on all five chromosomes. The previously identified tomato gene ER6 (Zegzouti et al., 1999) and the previously identified nodulation gene from Vicia faba, VfENOD18 (Hohnjec et al., 2000), encode members of the USP family. They have approximately 50% amino acid sequence similarity to OsUSP1. Overall, USP proteins are present in archaea, in bacteria and in plants, but not in animals.
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OsUSP1 belongs to the ATP-binding MJ0577 subfamily of USP proteins
Based on structural and biochemical data it was predicted that the universal stress protein family in bacteria segregates into two groups, a group that binds ATP and a group that does not bind ATP (Sousa and McKay, 2001). The ATP-binding Usp proteins are represented by the MJ0577 protein from Methanococcus jannaschii (Zarembinski et al., 1998) whereas the USP proteins which do not bind ATP are represented by the UspA proteins from Haemophilus influenzae and Escherichia coli. Both, MJ0577 from M. jannaschii and UspA from H. influenzae were shown to exist as homodimers of similar structure (Sousa and McKay, 2001; Zarembinski et al., 1998).
For M. jannaschii MJ0577 the residues which are in contact with ATP were identified by crystal structure analysis (Zarembinski et al., 1998). These domains are indicated in Fig. 3A. Comparison between UspAs from E.coli and H. influenzae, MJ0577 from M. jannaschii and several plant Usp homologues revealed that the ATP-binding regions are highly conserved between MJ0577 and OsUSP1 from rice, ER6 from tomato and VfENOD18 from bean, but are less conserved in the UspA proteins from E.coli or H. influenzae. Zarembinski et al. (1998) also identified the residues which are present at the interface of the homodimer that is formed by bacterial Usp proteins. Sequence comparison indicated conservation between plant and bacterial Usp proteins within the dimerization domain (Fig. 3A). Using computational analysis the secondary structure of rice OsUSP1 was predicted. The distribution of 
-helices and ß-strands appeared to be very similar to that described for M. jannaschii MJ0577 as derived from crystal structure analysis (Zarembinski et al., 1998) supporting a close structural and, possibly, functional relationship between the two proteins (Fig. 3A). A similar secondary structure was also predicted for bean VfENOD18 (Becker et al., 2001).
Phylogenetic analysis of approximately 100 Usp-like proteins from plants and microbes further supported the conclusion that plant Usp homologues are related closer to the bacterial ATP-binding MJ0577 subfamily than to the bacterial UspA subfamily (data not shown). This relationship is presented in the simplified phylogenetic tree shown in Fig. 3B. For VfENOD18, it was shown experimentally that this protein can, in fact, bind ATP, confirming the phylogenetic relationship analysis (Becker et al., 2001).
OsUsp1 is regulated by ethylene
As described earlier, submergence induces a number of adaptations to hypoxic stress which are mediated by ethylene. In order to find out if the submergence induction of OsUsp1 expression was mediated by ethylene, excised stem sections containing the youngest internode were used and treated without an effector as control or with the ethylene-releasing compound ethephon (Fig. 4A, B). In stem sections that were incubated without the hormone a weak transient decline in OsUsp1 mRNA was seen after 2 h and 3 h of treatment. The lower mRNA level at 0 h might be due to weaker loading of the gel. Transcript levels recovered and remained constant until the end of the measuring period after 18 h. Ethephon application resulted in very strong OsUsp1 transcript accumulation within 1 h (Fig. 4B). The mRNA declined again to basal levels between 2 h and 3 h after ethephon addition and remained low up to the end of the treatment period. Thus, ethylene regulated OsUsp1 expression rapidly, strongly and transiently. A rough estimate from Fig. 4B suggested a half-life for OsUsp1 mRNA of 15 min or less. The time-course of gene induction in ethephon-treated stem sections closely resembled that observed in submerged plants (Figs 1, 4B).
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In another experiment, it was possible to show that ethylene induction was suppressed in the presence of norbornadiene, an inhibitor that competitively blocks binding of ethylene to its receptor (Fig. 4C). NBD by itself had no effect on OsUsp1 expression. The result thus indicated that ethylene signalling must take place for OsUsp1 to be induced.
To analyse whether OsUsp1 transcript accumulation in response to ethylene application was an immediate early ethylene response, stem sections were treated with cycloheximide in addition to ethephon. In the presence of the protein synthesis inhibitor OsUsp1 induction was abolished (data not shown) indicating that OsUsp1 is not a primary ethylene response gene, but rather requires protein synthesis in order to be induced.
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Discussion |
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In a differential screening for submergence-induced genes in rice, a plant homologue of the bacterial universal stress protein family was identified. UspA from Escherichia coli is induced by conditions that cause growth arrest and was suggested to modulate or reorganize the carbon flow during growth arrest (Nystrom and Neidhardt, 1994, 1996) and to defend against DNA damage (Gustavsson et al., 2002). Deletion of UspA resulted in sensitivity to UV exposure while overproduction in Escherichia coli reduced the growth rate in glucose minimal medium, but not in full medium. It also delayed the recovery from glucose starvation, an effect that was especially pronounced in cells that were starved anaerobically (Nystrom and Neidhardt, 1996).
Bacterial Usp proteins exist as ATP-binding proteins of the M. jannaschii MJ0577 type and as proteins of the H. influenzae UspA type that do not bind ATP. The ATP-binding motifs and their spacings are different in MJ0577 than in other known ATP-binding proteins (Zarembinski et al., 1998). Plant Usp homologues including OsUSP1 are most closely related to the MJ0577 group. Conservation of the ATP-interacting domains and conservation of the dimerization domain indicated that plant Usp proteins might exist as ATP-binding dimers. Using computer modelling, it was recently shown that the Usp homologue VfENOD18 from bean has a highly similar secondary structure prediction to MJ0577. It was further shown that VfENOD18 is an ATP-binding protein (Becker et al., 2001).
The ATP bound to MJ0577 was not hydrolysed during purification (Zarembinski et al., 1998). It was therefore assumed that one or more additional factors are required for hydrolysis. This assumption was supported by the observation that purified MJ0577 had ATPase activity when incubated in an M. jannaschii cell extract. Based on these observations, it was proposed that MJ0577 acts as a molecular switch. The observed rapid and strong OsUsp1 induction may indicate that OsUSP1 is required early and transiently and may thus function as a switch in submergence stress adaptation. However, no evidence exists in bacteria or plants as to the molecular or biochemical mode of action of such a switch.
A database search indicated that OsUSP1 homologues are ubiquitous in plants and that they are encoded by gene families. However, to date, only two other members of this plant protein family have been described. In no case has a function been identified. The ER6 gene from tomato was induced during tomato fruit ripening. The highest transcript levels were detected in red fruit. In late immature green fruit, but not in leaves or roots of tomato plants, ER6 gene induction by ethylene was observed (Zegzouti et al., 1999). VfENOD18 is a nodulation gene that is exclusively expressed in zone III of root nodules in Vicia faba (Hohnjec et al., 2000). Zone III is the nitrogen-fixing zone and is characterized by highly active carbon and nitrogen metabolism (Pawlowski, 1997) and by lack of oxygen (Soupène et al., 1995). Hypoxia is a common feature of submerged rice plants and of zone III of bean root nodules. Ethylene plays an important role in tomato fruit ripening and in the submergence adaptation of semi-aquatic plants such as rice. In submergence adaptation, the growth response of the internode involves abscisic acid and gibberellic acid in addition to ethylene (Kende et al., 1998). However, ethylene regulation of OsUsp1 occurs not only in the growing region where ABA and GA act, but also in differentiated tissues. Nonetheless, involvement of ABA and GA in OsUsp1 regulation needs to be investigated. Even though ethylene has been implicated in the early steps of root nodulation, a role for ethylene in mature root nodules has not been described ((Fernández-López et al., 1998; Morris and Djordjevic, 2001; Oldroyd et al., 2001; Pawlowski, 1997). Instead, complete lack of oxygen as found in mature root nodules prevents ethylene biosynthesis. Thus, a role for ethylene in VfENOD18 gene regulation cannot be assumed.
On the other hand, ripening tomato fruit, hypoxic internodal tissue and root nodules are characterized by a change in metabolic activity to serve the particular tasks of the respective plant organ. Metabolic adaptation under severe conditions might, therefore, be seen as a common feature that correlates with Usp expression in bean nodules, tomato fruit and in the rice internode. This adaptive process may be mediated by ethylene or by other stress signals. It remains to be established what the precise function and the mode of action of these novel proteins are.
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