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J. Biol. Chem., Vol. 275, Issue 49, 38467-38473, December 8, 2000

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An Early Ethylene Up-regulated Gene Encoding a Calmodulin-binding Protein Involved in Plant Senescence and Death^*

Tianbao Yang and B. W. Poovaiah

From the Laboratory of Plant Molecular Biology and Physiology, Department of Horticulture, Washington State University, Pullman, Washington 99164-6414

Received for publication, April 26, 2000, and in revised form, July 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

³⁵S-Labeled calmodulin (CaM) was used to screen a tobacco anther cDNA library. A positive clone (NtER1) with high homology to an early ethylene-up-regulated gene (ER66) in tomato, and an Arabidopsis homolog was isolated and characterized. Based on the helical wheel projection, a 25-mer peptide corresponding to the predicted CaM-binding region of NtER1 (amino acids 796-820) was synthesized. The gel-mobility shift assay showed that the peptide formed a stable complex with CaM only in the presence of Ca²⁺. CaM binds to NtER1 with high affinity (K_d ~ 12 nM) in a calcium-dependent manner. Tobacco flowers at different stages of development were treated with ethylene or with 1-methylcyclopropene for 2 h before treating with ethylene. Northern analysis showed that the NtER1 was rapidly induced after 15 min of exposure to ethylene. However, the 2-h 1-methylcyclopropene treatment totally blocked NtER1 expression in flowers at all stages of development, suggesting that NtER1 is an early ethylene-up-regulated gene. The senescing leaves and petals had significantly increased NtER1 induction as compared with young leaves and petals, implying that NtER1 is developmentally regulated and acts as a trigger for senescence and death. This is the first documented evidence for the involvement of Ca²⁺/CaM-mediated signaling in ethylene action.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In plants, like in animals, Ca²⁺ is known to act as a mediator of stimulus-response coupling in the regulation of diverse cellular functions which are triggered by a variety of hormonal and environmental stimuli (1-4). Calmodulin (CaM),¹ a ubiquitous Ca²⁺-binding protein in eukaryotes is the primary intracellular Ca²⁺ receptor, which transduces the second messenger Ca²⁺ signal by binding to and altering the activity of the CaM-binding proteins, thereby generating the physiological responses to stimuli (3-7). In recent years, an increasing number of CaM-binding proteins in plants have been reported (6, 7). There has been considerable interest in identifying and characterizing these proteins in order to understand the role of Ca²⁺/CaM-mediated signal transduction in response to various environmental and hormonal stimuli.

The gaseous hormone ethylene has profound effects on plant growth and development. There are numerous responses to ethylene throughout plant development, including induction of ripening in climacteric fruits, senescence, programmed cell death, abscission of various organs, promotion of seed germination, promotion, or inhibition of flowering (8). The ethylene biosynthetic pathway and aspects of its regulation have been determined (9, 10). Environmental stresses such as wounding, pathogen attack, and flooding can induce ethylene production; the stress-induced ethylene in turn can lead to certain defense responses such as accelerated senescence, abscission of infected organs, or induction of specific defense proteins (8). The physiological responses to ethylene are complex, and not much is known about the mechanism of ethylene action (10). However, progress is being made in the understanding of the ethylene signal perception and transduction mainly on the basis of the isolation of ethylene-responsive mutants (11-14). The identification of early ethylene-regulated genes is another strategy in gaining further understanding of the molecular mechanisms of ethylene action (15, 16).

There is a close relationship between cell calcium status and ethylene. For example, calcium can influence ethylene biosynthesis by affecting 1-aminocyclopropane-1-carboxylic acid oxidase transcription (17) and activity (18). Raz and Fluhr (19) reported that calcium is essential for the ethylene-dependent pathway of pathogenesis-related protein induction, and direct elevation of cytosolic calcium induced chitinase accumulation. Calcium is also required for ethylene-promoted morphogenic responses in seedlings (19). However, the molecular mechanism of Ca²⁺ and Ca²⁺/CaM in ethylene action is not clear. Using the ³⁵S-labeled CaM-binding approach, we isolated and characterized a tobacco gene, NtER1, which has high homology with tomato ethylene-up-regulated gene ER66 (16). Results described here demonstrate that NtER1 is an early ethylene-responsive gene, and CaM binds to NtER1 protein only in the presence of calcium. Furthermore, our results also indicate that Ca²⁺/CaM is involved in the mechanism of ethylene action.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of ³⁵S-Labeled CaM-- ³⁵S-Labeled recombinant CaM was prepared by using a potato CaM (PCM6) cloned into the pET-3b expression vector (20, 21). The specificity of ³⁵S-labeled CaM was about 0.5 × 10⁶ cpm/µg.

Screening the cDNA Library and DNA Sequence Analysis-- A tobacco anther cDNA expression library (ZAP II) prepared in our laboratory was screened using ³⁵S-labeled PCM6 as described (21). The positive cDNA clones were sequenced on both strands. DNA sequences were analyzed using GCG versions 8.0 and 9.0 software (22).

Plant Materials and Treatments-- Nicotiana tabacum cv. Xanthi plants grown under standard greenhouse conditions were used in this investigation. For ethylene treatment, flowers at various stages of development were excised and treated in 250-ml sealed jars with 100 µl liter¹ of ethylene for 15 min to 24 h at room temperature. The control flowers were exposed to air alone. For the 1-methylcyclopropene (MCP) treatment, tobacco flowers were treated for 2 h with 100 µl liter¹ MCP prior to the ethylene treatment. After treatments, the flowers were immediately frozen in liquid nitrogen and stored at 80 °C until RNA extraction.

RNA Isolation and Northern Analysis-- Total RNA was isolated from frozen tissue essentially as described (23). RNA samples (50 µg) were denatured and separated on 1.5% formaldehyde/agarose gels. Following transfer to Hybond N⁺ filters, the blots were hybridized using the ³²P-labeled NtER1 cDNA fragment and washed as described earlier (24). Blots were stripped and reprobed with a fragment of Arabidopsis 18 S rDNA (accession no. X16077, nucleotides 158-1669). The blots were exposed on the Kodak x-ray films from 1 to 4 days depending on the intensity of the signals.

Construction of DNA Templates Coding NtER1 Proteins-- The template coding for C-terminal 643 amino acids of NtER1 was produced by polymerase chain reaction amplification from the original cDNA with NtER1 specific oligonucleotides containing appropriate restriction sites (NdeI at the 5' end, and BamHI at the 3' end) for cloning into the downstream of His₆ tag into the pET-14b expression vector (Novagen, Inc.). The NtER1 was expressed in Escherichia coli strain BL21(DE3) pLysS according to the method of Studier et al. (25). The nucleotide sequence of the cloned fragment derived by polymerase chain reaction amplification was determined after cloning into the pET-14b vector, using oligonucleotides designed for sequencing from both sides of the pET-14b cloning sites as primers.

³⁵S-Recombinant CaM Binding Assay-- The NtER1 protein was extracted, and purified essentially as described (21). The amount of protein was estimated by the Bradford's method (26) using a protein assay kit (Bio-Rad). Proteins were separated by SDS-polyacrylamide gel electophoresis (SDS-PAGE), electrotransferred onto polyvinylidene difluoride membrane (Millipore), and incubated with ³⁵S-labeled recombinant CaM with 1 mM CaCl₂ or 2 mM EGTA as described (21). The membrane was washed with 25 mM Tris-HCl, pH 7.5, and 1 mM CaCl₂ or 2 mM EGTA and exposed to the x-ray film overnight.

Peptide Binding to CaM-- The synthetic peptides were prepared using Applied Biosystems peptide synthesizer 431A in the Laboratory of Bioanalysis and Biotechnology, Washington State University. Samples containing 240 pmol (4 µg) of bovine CaM (Sigma) and different amounts of purified synthetic peptides in 100 mM Tris-HCl, pH 7.2, and either 1 mM CaCl₂ or 2 mM EGTA in a total volume of 30 µl were incubated for 1 h at room temperature. The samples were analyzed by nondenaturing PAGE as described (27).

CaM Binding Affinity Assay-- The purified recombinant NtER1 (4 pmol) was spotted on an Immobilion membrane (Millipore) and incubated with 0, 5, 10, 20, 40, 60, 100, and 500 nM ³⁵S-labeled CaM with 1 mM CaCl₂ overnight. After washing in 25 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 1 mM CaCl₂, radioactivities of bound CaM on each filter and free CaM in the incubation buffer collected before washing were measured using a liquid scintillation counter. In order to eliminate the nonspecific CaM binding, the BSA was used as a negative control. The average background count was subtracted from the counts of NtER1 protein samples when calculating the specific binding. The dissociation constant K_d was calculated based on Scatchard plot.

Southern Blot Analysis-- Tobacco genomic DNA was extracted as described (28). Ten µg of DNA was digested with BamHI, BglII, and SacI; separated by electrophoresis on 0.8% agarose Tris acetate gel; and transferred to Hybond N⁺ nylon membrane (Amersham Pharmacia Biotech) with 0.4 M NaOH. Southern blot analysis was carried out as described earlier (28) using an NtER1 cDNA as a probe.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning of NtER1 cDNA-- The CaM-binding screening approach was used to isolate the CaM-binding proteins from a tobacco anther cDNA expression library. Eight positive clones were obtained from 2 × 10⁵ recombinant phages. One of these clones had high affinity to CaM. The nucleotide sequence of the 2135-bp cDNA clone contained a partial coding region (672 amino acids) and 116 bp of 3'-untranslated region. The clone was named as NtER1 because the deduced polypeptide of this partial cDNA had high homology with tomato ER66, an ethylene-up-regulated gene (16). In order to get the full clone, the same cDNA library was screened with the partial NtER1 cDNA as a probe. The nucleotide sequence and the deduced amino acid sequence of NtER1 are shown in Fig. 1. The cDNA codes for a polypeptide of 926 amino acids flanked by a 17-bp untranslated region at the 5' end and 153-bp untranslated region with poly(A)⁺ tail at the 3' end. The calculated molecular mass and the isoelectric point of the NtER1 polypeptide were 104 kDa and 8.69, respectively. The polypeptide was a serine-rich protein with 101 Ser in 926 amino acid residues.

View larger version (83K): [in this window] [in a new window]   Fig. 1.   Nucleotide and deduced amino acid sequences of NtER1. The CaM-binding region is underlined.

Comparisons to known sequences in GenBank^TM showed that NtER1 had high homology with tomato ER66, LeER66, and an Arabidopsis homolog, AtER-like (accession number AC007168), particularly at the C-terminal portions. The C-terminal 643 amino acids of NtER1 are aligned in Fig. 2 with LeER66 and AtER-like. In this portion, the NtER1 protein is 91.5% similar and 89.2% identical with tomato ER66, 69.2% similar and 62% identical with an Arabidopsis homolog. NtER1 also displays some homology with the FIN21.9 clone (accession number AC002130; 47.1% similarity and 37.9% identity) and a transcription factor-like protein (accession number Z97340; 40.5% similarity and 31.9% identity) of Arabidopsis (data not shown).

View larger version (65K): [in this window] [in a new window]   Fig. 2.   Comparison of the deduced amino acid sequence of NtER1 to tomato ER66 (LeER66) and an Arabidopsis homolog (AtER-like). The CaM-binding region is underlined. The accession numbers of LeER66 and AtER-like are AF096260 and AC007168.

Binding of CaM to NtER1-- The partial NtER1 (C-terminal 643 amino acid) was expressed in E. coli, using the pET-14b expression vector. The recombinant protein was present mainly in the soluble fraction. The 73.7-kDa fusion protein (71.5-kDa NtER1 plus 2.2-kDa N-terminal His₆ tag) was purified by CaM affinity chromatography to near homogeneity as judged by SDS-PAGE (data not shown). Using CaM-binding assay, it was shown that ³⁵S-labeled CaM binds to NtER1 protein only in the presence of Ca²⁺ (Fig. 3A). After adding 2 mM EGTA, no CaM binding was observed, suggesting that CaM binding to NtER1 is calcium-dependent. In the control experiment, the proteins from E. coli transformed with only the pET-14b vector did not show any CaM binding (data not shown).

View larger version (34K): [in this window] [in a new window]   Fig. 3.   CaM binding to NtER1 recombinant protein. A, the NtER1 protein was expressed in E. coli, and the cell extract was subjected to SDS-PAGE. The proteins were transferred onto a polyvinylidene difluoride membrane and incubated with 35S-labeled CaM (50 nM) in the buffer containing either 2 mM EGTA or 1 mM CaCl2. The protein size markers are shown on the left. B, gel mobility shift assay showing CaM binding to the synthetic peptide, corresponding to the amino acids 796-820 in NtER1 (listed on the top). Increasing amounts of the peptide (peptide/CaM molar ratios indicated) were incubated with 240 pmol of bovine CaM with 1 mM CaCl2; samples were separated by nondenaturing PAGE. Arrows indicate the position of the free CaM and the CaM/peptide complex.

Most characterized CaM-binding proteins have a secondary structural feature, basic amphiphilic -helix, even though the amino acid residues in the CaM-binding region of these proteins are not conserved (27, 29-31). A helical wheel projection of the peptide sequences predicted that the amino acids 796-820 in the C-terminal domain of NtER1 have a CaM-binding domain feature. The amino acid residues 805-818 formed an -helix with a hydrophobic face (including two leucines, one isoleucine, and one valine) and a basic hydrophilic face (including four arginines and one lysine). Particularly, a hydrophobic residue, often Trp (here amino acid 809), embedded in a context of basic residues Lys or Arg (here amino acids 810 and 811), is a common feature in known CaM targets (27, 30, 31). A peptide with 25 residues corresponding to the putative CaM-binding region (amino acids 796-820) was incubated with bovine CaM, and the complex formation was assessed by nondenaturing PAGE in the presence or absence of Ca²⁺. The 25-mer peptide is capable of forming a stable complex with CaM in the presence of Ca²⁺ (Fig. 3B), but not in the absence of Ca²⁺ (data not shown). Without adding the peptide, a single CaM band was observed. As the peptide was added, another band of low mobility appeared, representing the peptide-CaM complex. When the ratio of peptide to CaM was equal, the CaM band disappeared, and the intensity of the peptide-CaM complex increased. At a peptide to CaM molar ratio of 1.5, no free CaM was detected. These observations indicate that the peptide binds to Ca²⁺/CaM with a 1:1 stoichiometry.

Interestingly, the amino acids at CaM-binding region (amino acids 796-820) of NtER1 has a very high homology with the counterparts of ER66 and AtER-like (Fig. 2). The amino acids 431-455 of LeER66 are exactly the same as NtER1 amino acids 796-820. The AtER-like amino acids 909-933 have just two conservative amino acid substitutions. Both putative CaM-binding regions in LeER66 and AtER-like can form a basic amphiphilic -helix, and contain the Trp-Arg-Arg motif, suggesting that this type of proteins in general are CaM-binding proteins.

To determine the binding affinity of CaM to NtER1, the 4-pmol recombinant NtER1 was blotted on a filter and incubated with the different concentrations of ³⁵S-labeled CaM in the presence of 1 mM CaCl₂. The labeled CaM binds to NtER1 with a saturation at ~100 nM (Fig. 4), indicating the presence of a saturable high affinity binding site in NtER1. From the Scatchard plot analysis of the saturation curve, the dissociation constant (K_d) of CaM for NtER1 was estimated to be about 12 nM. The binding of CaM to NtER1 was completely blocked in the presence of 2 mM EGTA. The Scatchard analysis also indicated that the C-terminal portion of NtER1 has a single CaM-binding site.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Saturation curve of 35S-CaM binding to purified NtER1. E. coli-expressed NtER1 protein (4 pmol) was spotted on an membrane (Millipore) and incubated with different amounts of 35S-labeled CaM. Radioactivity of 35S-CaM bound in the filter and free CaM in the solution was measured using a liquid scintillation counter. Each measurement was the average of three repeats. The amount of bound CaM at each point was represented as a percentage of the maximal binding. The inset shows a Scatchard plot of data indicating that binding ratio of CaM to NtER1 is 1:1. Bound/free and bound CaM are expressed as B/F and B, respectively.

Expression of NtER1 in Response to Ethylene and during Senescence-- Tomato ER66 is an early ethylene-up-regulated gene (16). In order to confirm that NtER1 is regulated by ethylene, tobacco buds and fully opened flowers were treated with 100 µl liter¹ ethylene for 15 min to 24 h. The total RNA was isolated from plant tissues, and the expression of NtER1 mRNA was analyzed using Northern hybridization. A size of ~3.2-kilobase band was detected on the blot. Without ethylene application, NtER1 expression was detected in the fully opened flowers (Fig. 5A, lane 2). However, very low expression of NtER1 in the buds was detected after a longer exposure of the blot to x-ray film (data not shown). By treating with 100 µl liter¹ ethylene, the expression of NtER1 was quickly induced. The induction began within 15 min (Fig. 5A, lanes 3 and 4). Increased induction of NtER1 was observed after 5 and 24 h of ethylene treatment (Fig. 5A, lanes 5-8). Both buds and fully opened flowers showed increased expression following ethylene treatment, but the expression was higher in fully opened flowers in comparison to the buds. It is known that MCP is a potent inhibitor of ethylene action by blocking the ethylene receptor (32). Treatment with MCP prior to ethylene treatment blocked the induction of NtER1 (Fig. 5B). These results suggest that NtER1 is an early ethylene-up-regulated gene.

View larger version (79K): [in this window] [in a new window]   Fig. 5.   Effect of ethylene on expression of NtER1. A, the excised buds (B) and fully opened flowers (O) were treated with 100 ml liter1 ethylene with the time periods indicated on the top. B, prior to the ethylene treatment, tobacco flowers were treated with for 2 h with 100 µl liter1 MCP. The total RNAs were subjected to Northern analysis. Autoradiograms of Hybond N+ filter hybridized successively with 32P-labeled NtER1 cDNA and 18 S rDNA fragment 158-1669 (accession number X16077). Exposure time on x-ray film for the blot was 24 h.

The differential expression of NtER1 in the buds and fully opened flowers suggests that the NtER1 is associated with senescence, a form of programmed cell death. To investigate the possible role of NtER1 in senescence and aging, the levels of NtER1 mRNA were studied at different stages of development in tobacco petals and leaves. These organs are commonly used for studying plant senescence and programmed cell death. Fig. 6 shows that the NtER1 expression increases during senescence in both leaves and petals. In the immature and fully mature leaves, NtER1 expression was low (Fig. 6, lanes 1 and 2), while NtER1 was highly induced when the leaves started to turn yellow (Fig. 6, lane 3). In petals, NtER1 was detected at low levels in buds (Fig. 6, lane 4). There was a significant increase in the expression of NtER1 in petals of fully opened flowers coinciding with pollination and fertilization (Fig. 6, lane 5). As the flowers became senescent, NtER1 expression was further increased (Fig. 6, lane 6). Results shown in Fig. 6 also indicated that NtER1 expression was higher in petals as compared with leaves.

View larger version (50K): [in this window] [in a new window]   Fig. 6.   Expression of NtER1 in tobacco leaves and flower petals. The total RNAs from the immature, fully mature and senescing leaves and petals from bud, fully opened flowers, and senescing flowers were subjected to Northern analysis. Autoradiograms of Hybond N+ filter hybridized successively with 32P-labeled NtER1 cDNA and 18 S rDNA fragment 158-1669 (accession number X16077). Exposure time on x-ray film for the blot was 60 h.

Genomic Southern Blot Analysis-- Southern analysis was carried out using a genomic DNA extracted from tobacco leaves. DNA was digested with BamHI, BglII, and SacI, for which there was no restriction site in the NtER1 cDNA. The hybridization was performed with the NtER1 cDNA as a probe. Under high stringency conditions, one or two strong bands were observed after restriction with all three enzymes (Fig. 7). These results suggest that NtER1 belongs to a small gene family.

View larger version (79K): [in this window] [in a new window]   Fig. 7.   Southern blot analysis. The blot of tobacco genomic DNA digested with the restriction enzymes as indicated was hybridized with 32P-labeled NtER1 cDNA fragment. The sizes of DNA markers (kilobase pairs) are shown on the left, and the restriction enzymes on the top of the blot. Ba, BamHI; Bg, BglII; S, SacI.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Calmodulin is known to regulate a large number of enzymes in animals. In recent years, plant scientists have begun to isolate and characterize CaM-binding proteins to understand Ca²⁺/CaM-mediated signaling in plants (3-7). This information has increased the understanding of how calcium and CaM regulate the various biochemical and molecular processes that eventually lead to a physiological response.

In this study, we identified a novel CaM-binding protein encoded by NtER1, which has high homology to the tomato early ethylene-up-regulated gene ER66 (Fig. 2). Further investigations confirmed that NtER1 is up-regulated by ethylene (Fig. 5A) and MCP, which blocks ethylene action, also blocked the expression of NtER1. The expression of NtER1 is developmentally regulated, especially during senescence and aging. In the aging tissues, such as the yellow leaves and senescing petals, NtER1 expression is highly induced (Fig. 6). In other report, the expression of tomato ER66 is higher in the fruits at red stage and less at the mature green stage (16). The plant growth hormone ethylene, known as "death hormone," promotes aging, senescence, ripening, and wilting. A close relationship exists between plant senescence and ethylene biosynthesis (33, 34). In senescing tissues, ethylene biosynthesis increases (33). In bean explants, the senescence was deferred when ethylene synthesis was inhibited (35). Results from this investigation revealed that NtER1 expression greatly increased as the leaf and petal started to senesce (Fig. 6). This suggests that NtER1 is regulated by ethylene and it plays an important role in the progression of senescence and death. However, it is not known whether NtER1 is involved in other ethylene-regulated physiological processes, such as retardation of cell elongation, response to various stresses and defense responses.

A large number of studies have shown that plant senescence is associated with an elevated level of intracellular Ca²⁺ (36-39). Elevation of cytosolic Ca²⁺ in plants has been linked to cell death, including hypersensitive response cell death, tracheary cell death, aleorone cell death, leaf senescence, and petal senescence (40-46). The application of Ca²⁺ increases both DNase activity and DNA laddering in both animal cells and plant cells (47, 48). Results from this study show that the ethylene-up-regulated and senescence-associated gene, NtER1, encodes a Ca²⁺/CaM-binding protein (Fig. 3), and these results will increase the understanding of the role for calcium in plant senescence and cell death.

The response of NtER1 expression to ethylene is very rapid, within 15 min (Fig. 5A). The isolation and characterization of early ethylene-regulated genes are important in gaining an understanding of the molecular mechanisms of ethylene action and in defining the role of this hormone in various physiological processes in plants. Screening ethylene-treated mature green tomato fruit has led to the isolation of a set of ethylene regulated genes including the genes' homology to transcriptional co-activator MBF-1, elongation factor EF-Ts, stress-related protein catalase, and a protease inhibitor (16, 49). Others include a chitinase, which is ethylene-regulated in bean leaves (50, 51); a glutathione S-transferase, which responds to ethylene in senescing carnation petals (52); ER43, a putative GTP-binding protein; and ER50, a CTR-like clone (16). At present it is not known whether any of these proteins is regulated by Ca²⁺/CaM.

Identification of NtER1 and other related proteins such as tomato ER66 as CaM-binding proteins are significant in our understanding of the cross-talk between Ca²⁺/CaM-mediated signaling and ethylene action, as well as the molecular mechanisms of these signal transduction processes during senescence. It is known that ethylene and other plant hormones such as auxin, cytokinin, gibberellic acid, and abscisic acid can induce changes in free calcium levels in plant cells and calcium can act as a messenger in triggering various physiological processes. It is also known that calcium can affect ethylene biosynthesis by affecting the transcription and activity of 1-aminocyclopropane-1-carboxylic acid oxidase, a key enzyme in ethylene biosynthetic pathway (17, 18). Calcium also regulates the ethylene-dependent pathway of pathogenesis-related protein induction, and is required for ethylene-promoted morphogenic responses in seedlings (19). The results described here indicate that Ca²⁺/CaM interacts with NtER1 protein, which is involved in ethylene action leading to senescence and death. It is possible to control the onset of senescence by manipulating the expression of NtER1 gene. Delay of senescence could have major implications in prolonging the storage life of various agricultural products, especially fruits, vegetables, and flowers.

In summary, the sequence of events that leads to programmed senescence and death involves two crucial steps. The first step involves the biosynthesis of ethylene, and the second step involves the increase of cytosolic calcium and the induction of NtER1. This study suggests that Ca²⁺/CaM binds to NtER1 protein, and the complex then triggers a cascade of events that ultimately lead to senescence and death. Our earlier study indicated that Ca²⁺/CaM is involved in auxin action by binding to SAUR (small auxin up RNA) proteins (31). Identifying the early ethylene-regulated NtER1 as a CaM-binding protein further strengthens the importance of Ca²⁺/CaM messenger system in plant hormone action. It is quite clear that Ca²⁺/CaM affect gibberellic acid- and abscisic acid-regulated seed germination (53). Further isolation and characterization of the Ca²⁺/CaM-regulated, hormone-responsive proteins should help in the understanding of hormonal control of plant growth and development.

	ACKNOWLEDGEMENT

We are grateful to Dr. John Fellman, David R. Rudell, and Scott Mattinson for help in ethylene and MCP treatments of tobacco flowers.

	FOOTNOTES

^* This work was supported in part by National Science Foundation Grant MCB-0082256 and National Aeronautics and Space Administration Grant NAG-10-0061.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF253511.

To whom correspondence should be addressed. Tel.: 509-335-2487; Fax: 509-335-8690; E-mail: poovaiah@wsu.edu.

Published, JBC Papers in Press, August 21, /2000, DOI 10.1074/jbc.M003566200

	ABBREVIATIONS

The abbreviations used are: CaM, calmodulin; MCP, 1-methylcyclopropene; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

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				Y.-F. Chen, M. D. Randlett, J. L. Findell, and G. E. Schaller Localization of the Ethylene Receptor ETR1 to the Endoplasmic Reticulum of Arabidopsis J. Biol. Chem., May 24, 2002; 277(22): 19861 - 19866. [Abstract] [Full Text] [PDF]

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