Regulation of Senescence-Related Gene Expression in ... - NCBI

Received for publication December 11, 1989 Accepted March 15, 1990

Plant Physiol. (1990) 93, 1370-1375 0032-0889/90/93/1 370/06/$01 .00/0

Regulation of Senescence-Related Gene Expression in Carnation Flower Petals by Ethylene1 Kay A. Lawton, Kashchandra G. Raghothama, Peter B. Goldsbrough, and William R. Woodson* Department of Horticulture, Purdue University, West Lafayette, Indiana 47907 morphological symptom of senescence. Inhibitors of ethylene biosynthesis or action delay the onset of senescence as well as the associated increase in ethylene biosynthesis. Exposing preclimacteric flowers to exogenous ethylene induces premature petal senescence and an autoenhancement of ethylene production. Flower senescence in carnation is associated with changes in gene expression involving both protein and mRNA changes (29). Some of these changes relate temporally to the onset of increased ethylene biosynthesis and are the result of increased expression of specific senescence-related mRNAs (14). Accumulation of some of these senescence-related mRNAs can be prevented by inhibitors of ethylene biosynthesis or action (14, 30). Given the strict requirement for perception of ethylene in the induction of petal senescence, the role of ethylene in the regulation of senescence-related genes is of interest. To begin to understand the mechanism of ethylene action during flower senescence, we examined the response of three cloned petal senescence-related mRNAs to ethylene. We show that continued perception of ethylene is required for senescence-related gene expression. Furthermore, changes in senescence-related gene expression are related to changes in transcription rate, ethylene concentration, and tissue responsiveness to ethylene. Our results indicate ethylene modulates senescence-related gene expression in carnation petals by a variety of mechanisms.

ABSTRACT Ethylene plays a regulatory role in camation (Dianthus caryophyllus L.) flower senescence. Petal senescence coincides with a burst of ethylene production, is induced prematurely in response to exogenous ethylene, and is delayed by inhibitors of ethylene biosynthesis or action. We have investigated the role of ethylene in the regulation of three senescence-related cDNA clones isolated from a senescent camation petal library (KA Lawton et al. [1989] Plant Physiol 90: 690-696). Expression of two of the cloned mRNAs in response to ethylene is floral specific, while the expression of another mRNA can be induced in both leaves and flowers exposed to ethylene. Although ethylene induces expression of these mRNAs in petals, message abundance decreases when flowers are removed from ethylene unless an autoenhancement of ethylene production is induced. This indicates continued perception of ethylene is required for their expression. Interruption of ethylene action following the onset of natural senescence results in a substantial decrease in transcript abundance of two of these mRNAs. However, the abundance of another mRNA remains unaffected, indicating this gene responds to temporal cues as well as to ethylene. As flowers age the dosage of exogenous ethylene required to induce expression of the cloned mRNAs decreases, indicating sensitivity to ethylene changes as the tissue matures. Nuclear run-on transcription experiments indicate that relative transcription rates of cloned mRNAs increase in response to exogenous ethylene.

MATERIALS AND METHODS Plant Material

Ethylene plays a regulatory role in many processes of plant growth and development. Among these are seed germination, seedling growth, leaf and root growth, abscission, plant senescence, fruit development, and plant response to stress (1). For several of these processes, this regulation has been shown to include the expression of specific genes. During the ripening of climacteric fruits such as tomato and avocado, ethylene modulates the expression of several ripening-specific genes (8, 15). Ethylene also regulates expression of cellulase (26) and chitinase (13) during bean leaf abscission. In addition, plant defense genes have been shown to be regulated by ethylene during the wounding response (1 1). The regulatory role of ethylene in carnation flower senescence is well established (4). A climacteric increase in ethylene production coincides with petal inrolling, the first visible

Carnation (Dianthus caryophyllus L. cv 'White Sim') flowers were harvested at anthesis from plants grown under greenhouse conditions and exposed to ethylene as described (30). Vegetative and reproductive tissues were collected, frozen immediately in liquid nitrogen, and stored at -70°C until used for RNA extraction.

Chemicals

a[32P]dCTP at >3000 Ci/mmol and a[32P]UTP at >3000 Ci/mmol were obtained from New England Nuclear. Nick translation kit was purchased from Amersham. All other chemicals and reagents were from Bethesda Research Laboratories or Sigma Chemical Co.

'Supported by grants from the National Science Foundation (DCB8911205) and the Indiana Corporation for Science and Technology. Publication No. 12,321 of the Purdue University Agricultural Experiment Station.

Ethylene Measurement Ethylene production was measured as previously described

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RNA Extraction and Analysis

Total RNA was extracted from frozen tissue as described elsewhere (29, 30). RNA was electrophoretically separated on 1% agarose gels containing 2.2 M formaldehyde and transferred to nitrocellulose. The nitrocellulose filter was prehybridized 4 h at 42TC in a solution containing 50% (v/v) formamide, 5X Denhart's (IX Denhart's is 0.02% PVP, 0.02% Ficoll, 0.02% BSA), 0.1% SDS, 6X SSPE (IX SSPE is 0.15 M NaCl, 10 mM NaH2PO4, 1 mm EDTA [pH 7.4]), and 100 gg/mL denatured salmon sperm DNA. Hybridization was carried out in identical buffer solution plus 5 X lIO cpm/ mL denatured 32P-labeled DNA probe for 18 h. Plasmids used for RNA hybridization analysis were cDNA clones pSR5, pSR8, and pSR12 (2500, 640, 2600 bp inserts, respectively) which correspond to mRNAs that are expressed in senescent carnation petals (14). Plasmid DNA was labeled by nick translation to a specific activity of about 2 X 108 cpm/,hg DNA. The filters were washed in 3X SSC (IX SSC is 0.15 M NaCl, 15 mm Na-citrate [pH 7.0]), 0.1 % SDS (w/v) for 4 h at 42TC, and then exposed to Kodak XAR-5 film with an intensifying screen at -70TC for 18 to 36 h. Total RNA was slot blotted according to manufacturers instructions using a Schleicher and Schuell Minifold II Slot Blot apparatus. Prehybridization and hybridization were carried out as described above for RNA-blot analysis. Nuclei Isolation and Nuclear Run-on Transcription Nuclei from 20 g carnation flower petals held in air or exposed to 10 ,L/L C2H4 for 12 h were isolated as described (16-18). An aliquot of nuclei was stained with 4,6-diamidino2-phenylindole and examined by fluorescence microscopy to determine integrity and quantity. [32P]nRNA synthesis was carried out at 30C for 20 min essentially as described by Walling et al. (27) except that 10,u/mL RNasin was included in the reaction. Nuclei from the 80% Percoll/2 M sucrose interface incorporated 87% of the [32P]UTP incorporated. Nuclei from this interface were used for all experiments. In the presence of 2 ,ug/mL a-amanitin [32P]UTP incorporation was inhibited by an average of 58%.

[32P]nRNA Isolation The transcription reaction was followed by treatment with 80 ug/mL DNase I for 10 min at room temperature. The mixture was adjusted to 5 mM Tris (pH 7.6), 0.5 mM EDTA, 0.5% SDS and digested with 8,ug/mL proteinase K for 1 h at 42°C. The RNA transcripts were extracted with phenol/chloroform/isoamyl-alcohol (25:24:1 v/v) twice and tRNA was added to a final concentration of 0.1 mg/mL as a carrier. RNA was precipitated in 1/20 volume 4 M sodium acetate (pH 6.0) and 2 volumes ethanol at -20°C overnight. The resulting pellet was resuspended in 10 mm sodium pyrophosphate and precipitated with 10% trichloroacetic acid. The RNA was washed with 95% ethanol and resuspended in hybridization buffer (10 mM Tes (N-tris[hydroxymethyl] methyl-2-aminoethane-sulfonic acid) (pH 7.4), 1 mm EDTA, 0.2% SDS, 300 mM NaCl, 30% formamide, 0.2 mg/mL tRNA).

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Hybridization of nRNA Transcripts Five ,g of plasmid DNA was denatured and slot blotted onto nitrocellulose using a Schleicher and Schuell Minifold II Slot Blot apparatus and following manufacturer's instructions. Prehybridization and hybridization were for 24 and 48 h, respectively, at 42°C. Each hybridization reaction mixture contained 5 X 106 cpm/mL. Following hybridization the filters were washed three times at room temperature for 30 min in 3X SSC, 0.1 % SDS. Filters were then treated with 2 ,ug/mL RNase for 30 min at 37C in 3X SSC, then washed once in 3X SSC for 30 min at 42°C and twice in 1X SSC at 40C for 30 min. The filters were dried and exposed to Kodak XAR-5 film with an intensifying screen at -70°C for 48 h. There was no detectable hybridization to vector sequences. RESULTS Expression of Senescence-Related Genes in Response to Transient Ethylene Exposure Exposure of preclimacteric flowers to ethylene induces the developmental program of petal senescence (4). We previously reported that exposing preclimacteric petals to 7.5 ttL/L ethylene for 3 h was sufficient to induce senescence-related gene expression (14). However, induction of premature petal senescence and ethylene synthesis requires ethylene exposure for a longer duration (30). To examine the effects of transient ethylene exposure on senescence-related mRNAs; precimacteric flowers were exposed to 10 AL/L ethylene for 3 or 12 h. Ethylene production was measured and RNA was extracted from flower petals immediately after removal from the ethylene atmosphere, as well as, from flowers that had been held in air for an additional 12h following ethylene exposure. Figure 1 shows a 12 h ethylene exposure is required to induce ethylene synthesis. Furthermore, ethylene biosynthesis continues in these flowers after 12 h in air. These flowers go on to senesce prematurely (data not shown). Gel blots of RNA extracted from ethylene treated flower petals are shown in Figure 2. Although a 3 h exposure to ethylene is sufficient to induce accumulation of senescence-related gene transcripts, the steady state message levels decrease when flowers are returned to air for an additional 12 h and the flowers do not senesce prematurely. However, when the duration of ethylene exposure is sufficient to induce ethylene synthesis, mRNA abundance remains elevated when flowers are returned to air for an additional 12 h. These results indicate continued perception of ethylene is required for continued senescencerelated gene expression. Effect of NBD2 on Senescence-Related Gene Expression in Climacteric Flower Petals Previously we have shown autoenhancement of ethylene production and petal senescence require continued ethylene perception since transferring climacteric flowers to the ethylene action inhibitor NBD results in decreased ethylene production and reversal of senescence symptoms (28). To determine the effect of interrupting ethylene action following the 2Abbreviation: NBD, 2,5-norbornadiene.

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concentration of exogenous ethylene to induce gene expression (Fig. 4). Each of the three senescence-related mRNAs exhibited a unique dose-response curve in response to the ethylene treatment. The mRNA represented by pSR5 appeared to be most responsive to ethylene, accumulating 59% of maximum mRNA abundance in response to 100 ,uL/L ethylene at the TB stage. Both pSR5 and pSR12 message abundance reached saturated steady state levels over a narrow, 2-order of magnitude range of ethylene concentration. In contrast, the mRNA represented by pSR8 exhibited a broader dose-response. This result suggests that there are differences in the way that ethylene regulates the expression of these genes.

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Figure 1. Effect of ethylene exposure on ethylene production in preclimacteric carnation petals. Carnation flowers were harvested when petals were 90° reflexed to calyx and exposed to 10 gL/L ethylene for 0, 3, or 12 h. The 3 h treatment was started 9 h after petals were harvested so that all petals were equal age from harvest when ethylene production was first measured. Rates of ethylene production were measured immediately upon removal from the ethylene atmosphere and after the flowers had been held in air for an additional 12 h.

onset of natural senescence on senescence-related mRNAs, climacteric petals were transferred to an atmosphere containing 2000 gL/L NBD. RNA was extracted at various times after transferring flowers to NBD and senescence-related transcript level was determined by RNA blot analysis (Fig. 3). Treatment of climacteric flowers with NBD for 6 h resulted in a substantial decrease in abundance of pSR8 and pSR12 transcripts. An additional 6 h incubation in NBD resulted in a further reduction of these transcripts. In contrast, the level of pSR5 mRNA remained elevated throughout the NBD treatment. This indicates continued ethylene synthesis and action are required for maintenance of high levels of pSR8 and pSR12 transcripts, whereas pSR5 mRNA is not affected.

Effect of Changes in Ethylene Responsiveness during Petal Development on Senescence-Related Gene Expression The induction of petal senescence by ethylene has been shown to be dependent both on ethylene concentration and stage of petal development (2). As petals age, a lower concentration of ethylene is required to elicit the response of petal senescence. To further investigate the role of ethylene responsiveness in carnation petals we examined the effect of ethylene concentration on senescence-related gene expression at various stages of flower development. Flowers were harvested at the tight bud (TB), expanding petal (EP), and open flower (OF) stages (30) and exposed to 10 ,L/L ethylene for 6 h. For each of the senescence-related mRNAs, an increase in petal age was associated with increased capacity for a given

Organ Specificitv of Ethylene-induced Senescence-Related Gene Expression Previously we showed expression of senescence-related mRNAs was limited to climacteric flower tissue with maximum mRNA abundance in climacteric petals (14). To determine if floral specificity of senescence-related gene expression was retained in response to exogenous ethylene, leaves and preclimacteric flowers were exposed to 10 ML/L ethylene for 12 h. Following ethylene treatment, RNA was extracted from leaves, styles, ovaries, and petals and senescence-related transcript level was assayed by RNA blot analysis. The results are shown in Figure 5. Exogenous ethylene application induced increased abundance of pSR5 mRNA in leaves as well as petals. In contrast, transcripts of pSR8 and pSR12 accumulate only in styles and petals in response to exogenous ethylene. This shows ethylene-inducible expression of pSR8 and pSR12 is specific to senescing flower tissue.

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Figure 2. Effect of transient ethylene exposure on abundance of senescence-related mRNAs in carnation petals. Preclimacteric (day 0) carnation flowers were exposed to 10 IAL/L ethylene for 0, 3, or 12 h. RNA was extracted from petal tissue immediately upon removal from ethylene atmosphere or after flowers had been held in air for an additional 12 h. Ten g of total RNA was separated electrophoretically under denaturing conditions in 1% agarose, blotted to nitrocellulose, and hybridized to U2P-labeled cDNA clones. Blots hybridized with probes from pSR5, pSR8, and pSR12 were exposed to x-ray film with an intensifying screen at -700C for 18, 36, or 18 h, respectively.


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cence (30). The question remains as to whether ethylene acts to trigger senescence or control the processes of a previously initiated developmental stage. Our results show that ethylene is required not only to induce senescence-related gene expression, but also to maintain the elevated mRNA levels in preclimacteric flowers (Fig. 2). Senescence-related transcript abundance declined in the absence of ethylene unless the exogenous ethylene exposure was sufficient to induce an autoenhancement of ethylene synthesis (Fig. 1). During natural senescence, the accumulation of pSR8 and pSR12 tran-

Figure 3. Effect of NBD on abundance of specific senescence-related mRNAs in climacteric flower petals. Climacteric flowers were exposed to 2000 ML/L NBD and RNA was extracted from petals at indicated times after transfer to an NBD atmosphere. Ten 9sg of total RNA was separated electrophoretically under denaturing conditions in 1% agarose, blotted to nitrocellulose, and hybridized with 32P-labeled cDNA clones. Blots of hybridizations were exposed to x-ray film with an intensifying screen at -700C for 18 h.

Changes in Rate of Senescence-Related Gene Transcription in Response to Ethylene Increased steady state transcript levels may reflect increased rates of gene transcription, enhanced posttranscriptional processing or increased mRNA stability. To determine whether transcriptional activation of these genes was responsible for ethylene-induced accumulation of senescence-related mRNAs, we isolated nuclei from preclimacteric petals that had been exposed to air or 10 ML/L ethylene for 12 h and used them to synthesize [32P]nRNA. The [32P]nRNA was isolated from nuclei, purified, and hybridized to cDNA clones immobilized on nitrocellulose. The transcriptional rates of all three genes increased substantially in ethylene treated petals (Fig. 6). This is in contrast to the constitutive transcription rate of rDNA. These results indicate ethylene induces increased transcription of senescence-related genes. This increased transcription accounts at least partially for increased senescence-related transcript accumulation in response to exogenous ethylene. DISCUSSION The maturation ofcarnation flower petals is associated with a substantial increase in the synthesis of ethylene which plays a role in regulating the developmental processes of senescence (4). Previously we reported the isolation ofthree cDNAs which represent senescence-related mRNAs from carnation petals (14). Here we have investigated the role of ethylene in the regulation of these cloned senescence-related mRNAs in an attempt to begin to understand the relationship between ethylene, gene expression, and flower senescence. Exposure of presenescent carnation flowers to exogenous ethylene induces the developmental program of petal senes-

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ETHYLENE (u/L) Figure 4. Densitometric scans of RNA gel blots hybridized to 32p_ labeled cDNA clones. Flowers at the tight bud (TB), expanding petal (EP), and open flower (OF) stages of development were exposed to increasing concentrations of ethylene for 6 h. Following ethylene exposure, RNA was extracted and 10 Mg of total RNA was slot blotted onto nitrocellulose and hybridized to pSR5, pSR8, and pSR12 32P-labeled plasmid DNA. Blots were exposed to x-ray film with an intensifying screen at -700C for 18 h. The resulting autoradiograms were scanned densitometrically, and peak areas are presented as percent of maximum hybridization for each probe.

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Figure 5. RNA-blot analysis of RNA isolated from various vegetative and floral organs of carnation held in air (A) or exposed to 10 ,1L/L ethylene for 12 h (E). Ten Mg of total RNA extracted from the various tissues was separated electrophoretically under denaturing conditions on 1% agarose, blotted to nitrocellulose, and hybridized to 32Plabeled cDNA clones. Blots were exposed to x-ray film with an intensifying screen at -700C for 18 h.

scripts occurs concomitant with the ethylene climacteric (14). It is clear from our data that continued perception of ethylene is required to maintain the elevated levels of these transcripts. Interruption of ethylene action by NBD following the onset of the ethylene climacteric results in a rapid decline in the abundance of pSR8 and pSR12 (Fig. 3). We have shown previously that this treatment also inhibits ethylene production and reverses the symptoms of senescence (28). In contrast, the level of pSR5 transcript remained high in climacteric tissue transferred to an atmosphere of NBD. It is apparent from these and previous results (14) that the accumulation of pSR5 transcript, while responsive to ethylene in precimacteric tissue, appears to be under the control of temporal signals independent of ethylene in mature petals. Several possibilities exist that can account for the different responses seen with pSR5 expression in preclimacteric and climacteric petals. First, it is possible that the NBD treatment was not completely effective in blocking ethylene action in climacteric flowers. However, this seems to be discounted by the effectiveness of this treatment on pSR8 and pSR12 transcript abundance. A second possibility is that the pSR5 transcript is very stable in comparison to other senescence-related genes. If this is the case, the stabilizing effect must be temporally regulated since the pSR5 transcript abundance declined in preclimacteric flowers following a transient ethylene treatment. Another possibility is that pSR5 mRNA accumulation is regulated by both ethylene and temporal factors. This regulation could be the result of the interaction of multiple regulatory elements and trans-acting factors controlling a single gene or the expression of different genes of a gene family. Future research will be directed at distinguishing between these possibilities. It has been proposed that the capacity of plant growth substances to exert control on plant development is dependent on tissue sensitivity to the substance (25), as well as hormone

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concentration. Although the molecular basis oftissue sensitivity is not known, it is generally believed to be dependent upon the presence and availability of specific receptor molecules. Presumably, the plant growth substance interacts with the receptor to form a hormone-receptor complex. The concentration and availability of this complex, in turn, mediates the plant response. Ethylene responses in plants are believed to involve the interaction of ethylene with a metalloprotein receptor (7). Ethylene binding has been demonstrated in carnation petals (23). Moreover, flower maturity is accompanied by an increase in tissue responsiveness to ethylene (4); however, this increase in responsiveness has not been related to an increase in ethylene binding capacity (6). Our results indicate the aging of flower petals is associated with an increase in the capacity for a given concentration of ethylene to induce the accumulation of senescence-related mRNAs. The dose-response curves for mRNA abundance (Fig. 4) indicate each of the three senescence-related transcripts show unique patterns of expression, with pSR5 exhibiting the maximum sensitivity to ethylene. Given an increase in responsiveness to ethylene with age, it is possible that the initial induction of senescence-related transcript abundance occurs in aging tissue in response to the change in tissue sensitivity. Ethylene has been shown to regulate expression of genes at the transcriptional as well as posttranscriptional levels (16, 20). We have determined ethylene exposure induces an increase in the transcription rate of these senescence-related genes. However, it is possible that mRNA abundance of these genes is also affected by improved mRNA stability or other posttranscriptional events. We are currently investigating these possibilities.

Figure 6. Nuclear run-on transcription of senescence-related genes in response to ethylene. Nuclei were isolated from petals held in air or exposed to 10 ML/L ethylene for 12 h and used for in vitro transcription reactions as described in "Materials and Methods." [32P] nRNA was isolated and hybridized to 5 jtg of pUC18, rDNA, pSR5, pSR8, and pSR1 2 DNA that had been linearized, denatured, and slotblotted to nitrocellulose. Autoradiograms were exposed at -700C for 24 h.


Examination of organ specific expression of senescencerelated mRNAs in response to ethylene resulted in two classes of mRNAs. Although maximum accumulation of all three cloned mRNAs was observed in petal tissue, the gene represented by pSR5 was also expressed in leaf tissue. In contrast, the transcripts of pSR8 and pSR12 remained floral specific, accumulating in styles exposed to exogenous ethylene. Interestingly, styles and petals are the floral organs that senesce in response to ethylene. Therefore, the protein products encoded by these genes may play a primary role in the senescence process.

Expression of genes in a tissue/organ-specific manner, during a particular phase of development and in response to a plant hormone, is presumed to be due to the interaction between specific DNA sequences 5' to the transcription start site and DNA binding factors present in the nucleus. Upstream DNA sequences that confer tissue-specific (12, 22), developmental (3), and hormonal (5, 19) regulation of gene expression have been identified for some genes. The specific interaction between DNA sequence elements and DNA binding factors during tomato fruit ripening (9, 10) and in response to the phytohormone gibberellin (21) has been demonstrated. The variable tissue specific, developmental, and ethyleneresponsive expression of these senescence-related genes makes them ideal subjects for elucidating the mechanisms by which DNA binding proteins interact with DNA sequence elements to regulate gene expression. We have identified genomic DNA clones for each of the cDNAs and are in the process of studying these characteristics. ACKNOWLEDGMENTS

We thank Drs. Avtar Handa and David Rhodes for their critical reading of the manuscript and Ms. Amanda Brandt for her excellent technical assistance.

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