JXB Advance Access originally published online on March 14, 2003
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Journal of Experimental Botany, Vol. 54, No. 386, pp. 1489-1490,
May 1, 2003
© 2003 Oxford University Press
Characterization of an ethylene receptor homologue from wheat and its expression during leaf senescence
Received 11 November 2002; Accepted 25 January 2003
Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
1 To whom correspondence should be addressed. Fax: +86 010 62590839. E-mail: mqh{at}ns.ibcas.ac.cn
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Abstract |
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A wheat ethylene receptor homologue (W-er1) was isolated from a wheat stem cDNA library using the Arabidopsis ETR1 cDNA as a probe. The predicted amino acid sequence of W-er1 is over 70% similar to ERS1 from Arabidopsis and exhibits homology to bacterial two-component response regulators within the histidine kinase domain. Northern hybridization demonstrated that W-er1 was expressed in stem, leaf and root tissues. Treatments known to induce senescence of detached leaves including jasmonate, abscisic acid and wounding, increased the accumulation of W-er1 mRNA, while benzyladenine treatment did not. These data suggest that W-er1 may play a role in the process of leaf senescence.
Key words: Ethylene receptor homologue, leaf senescence, wheat.
Ethylene is a simple gaseous plant hormone that regulates many diverse plant processes, ranging from seed germination to organ senescence. Although the biosynthetic pathway of ethylene is well understood, knowledge of the molecular mechanisms underlying ethylene signalling is limited. Genetic studies in Arabidopsis have provided evidence that ethylene perception in plants is mediated by a family of receptors including the ETR1, ETR2, ERS1, ERS2, and EIN4 gene products (Hua and Meyerowitz, 1998). These proteins are related to a superfamily of catalytic receptors prevalent in bacteria and some eukaryotic systems known as two-component regulators. Such regulators are typically composed of a sensor protein and a response regulator protein, which function together to regulate adaptive responses to a broad range of environmental stimuli (Sakakibara et al., 2000). After the initial cloning of ETR1 from Arabidopsis, ETR1-like genes have been reported from tomato, tobacco, pea, Rumex palustris, muskmelon, cucumber, passion fruit, mango fruit, and geranium. Although a basic framework of the initial events in the ethylene-response pathway is emerging from these studies, there are many issues that remain to be resolved. For example, the significance of different receptor isoforms is still unclear. Furthermore, the expression patterns of ethylene receptors from crop plants and, in particular, wheat, have not been fully investigated. In the present study, an ethylene-receptor homologue from wheat was cloned and its expression pattern was determined to begin to address its potential biological function.
Wheat plants (Triticum aestivum L. cv. H4564) were grown in a naturally lit glasshouse with normal irrigation and fertilization. Total RNA was isolated from wheat tissues by TRI reagent (Molecular Research Center, Inc, Cincinnati, USA) according to the manufacturer’s instructions. Poly(A)+ RNA was isolated using the PolyAT tractR mRNA Isolation Kit (Promega).
A wheat stem cDNA library was constructed using the ZAP-cDNA synthesis kit in conjunction with the Uni-ZAP unidirectional vector according to the manufacturer’s instructions (Stratagene). The library was screened using a 32P-labelled Arabidopsis ETR1 cDNA probe that was generated by RT-PCR using Arabidopsis stem poly(A)+ RNA as template and primers specific for the third and fourth exons of ETR1. The 5' primer was: 5'-GTGGCTGTAGC TCTCTCAC-3', and the 3' primer was: 5'-CTTGGAGATGGCGA GGCCAAG-3'. The RT-PCR product was sequenced to confirm its identity. The wheat ethylene receptor homologue (W-er1, accession no. AY082300 [GenBank] ) identified from this screen was found to be 1167 bp long with a 121 nucleotide 5'-untranslated region, and a 209 nucleotide 3'-untranslated region and poly(A)+ tail. An open reading frame (ORF) predicted to encode a 279 amino acid polypeptide was located between nucleotide positions 122 and 958.
The deduced protein sequence of W-er1 showed high similarity to ethylene receptors from other plants, including more than 70% similarity to a portion of the Arabidopsis ERS1 protein. In particular, four highly conserved motifs (LMQTILNISGNA, LKDTGCGIS, VFTKF, GSGLGL) are present in the W-er1 amino acid sequence (Fig. 1), which are proposed to be important for the histidine kinase activity of the two-component regulator. Structural analysis indicated that W-er1 lacks the sensor domain found in AtERS1 (data not shown). This same situation has been reported in tomato (Payton et al., 1996) and some prokaryotic systems. Many prokaryotic two-component signal transducers have their sensor and response regulators on separate proteins (Parkinson and Kofoid, 1992). The plant origin of W-er1 was confirmed by Southern blot hybridization (data not shown).
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Northern analysis was conducted to examine the gene expression pattern of W-er1 in various wheat organs. RNA hybridization signals were normalized using a soybean 18S ribosomal RNA probe. W-er1 mRNA was detected in leaves, stems and roots (Fig. 2), but the abundance in roots was a little lower as analysed by Phosphor Image and normalization relative to the 18S rRNA signal (data not shown). These data are consistent with that reported for AtERS1 of Arabidopsis (Hall et al., 2000) and LeETR1, LeETR2 and LeNR of tomato (Lashbrook et al., 1998), which demonstrated constitutive but variable expression of transcripts in different tissues.
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The expression of W-er1 in response to wounding, benzyladenine, abscisic acid, and methyl jasmonate was also investigated. Detached leaves of 10-d-old seedlings of wheat were wounded or incubated with benzyladenine (BA, 0.4 mM), abscisic acid (ABA, 0.1 mM) or methyl jasmonate (JA, 1 mM) and harvested at intervals up to 48 h. The wounding treatment was to cut the leaf in two and then incubate the ends in water. Incubations were carried out in a dish with wet paper in darkness at 26 °C. The amount of chlorophyll extracted from leaves was used as an indicator of leaf senescence. Leaves were extracted with acetone and chlorophyll content was measured by absorption at A663 and A645. For the control, an intact leaf from a wheat plant was used. Wounding, ABA and JA all induced leaf senescence as demonstrated by a reduction of chlorophyll in treated leaves (Table 1), with JA treatment showing the greatest effect (Wsternack and Parthier, 1997). W-er1 mRNA levels were determined for each leaf treatment by northern hybridization and normalized relative to the 18S rRNA signal (Table 2). The same treatments that induced leaf senescence (wounding, ABA and JA treatment) stimulated the accumulation of W-er1 mRNA, which suggests that W-er1 may play a role in the senescence of detached leaves. In comparison, BA treatment, which did not stimulate leaf senescence, had little influence on W-er1 mRNA levels.
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It is well known that plant hormones are involved in the regulation of leaf senescence (Quirino et al., 2000). In monocot plants like oat, rice and wheat, evidence suggests that jasmonate and ABA promote leaf senescence, while cytokinins delay senescence. The actions of ethylene, however, are not clear. The above data suggest that W-er1 is involved in the senescence process of detached leaves. Research is necessary to determine whether cross-talk between ethylene and other hormones like jasmonate and ABA may contribute to the process of leaf senescence.
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Acknowledgements |
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This work was supported by Innovation Project of Chinese Academy of Sciences. We wish to sincerely thank Dr Bettina Deavours (Plant Biology Division, The Samuel Roberts Noble Foundation, USA) for critical reading of the manuscript.
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