JXB Advance Access originally published online on August 30, 2005
Journal of Experimental Botany 2005 56(420):2733-2744; doi:10.1093/jxb/eri266
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RESEARCH PAPER |
Ethylene-sensitivity regulates proteolytic activity and cysteine protease gene expression in petunia corollas*
1Department of Horticulture and Crop Science, 1680 Madison Avenue, The Ohio State University OARDC, Wooster, OH 44691, USA
2Food and Crop Research, Palmerston North, New Zealand
3Department of Environmental Horticulture, University of Florida, Gainesville, FL 32611, USA
To whom correspondence should be addressed. Fax: +1 330 263 3887. E-mail: jones.1968{at}osu.edu
Received 12 April 2005; Accepted 14 July 2005
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Abstract |
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To investigate ethylene's role in petal senescence, a comparative analysis of age-related changes in total protein, protease activity, and the expression of nine cysteine protease genes in the corollas of ethylene-sensitive Petuniaxhybrida cv. Mitchell Diploid (MD) and ethylene-insensitive (35S:etr1-1; line 44568) transgenic petunias was conducted. The later stages of corolla senescence in MD flowers were associated with decreased fresh weight, decreased total protein, and increased proteolytic activity. Corolla senescence was delayed by approximately 8 d in etr-44568 transgenic petunias, and decreases in corolla fresh weight, protein content, and maximum proteolytic activity were similarly delayed. Protease inhibitor studies indicated that the majority of the protease activity in senescing petals was due to cysteine proteases. Nine cysteine proteases expressed in petals were subsequently identified. Northern blot analysis indicated that six of the nine cysteine proteases showed increased transcript abundance during petal senescence. One of these cysteine proteases, PhCP10, was detected only in senescing tissues. Expression of four of the senescence-associated cysteine proteases was delayed, but not prevented in etr-44568 flowers. The other two senescence associated cysteine proteases had high levels of transcript accumulation in etr-44568 corollas at 8 d after flower opening, when MD flowers were senescing. These patterns suggest that age-related factors, other than ethylene, were regulating the up-regulation of these genes during flower ageing. The delay in visible symptoms and biochemical and molecular indicators of senescence in ethylene-insensitive flowers is consistent with the concept that ethylene modulates the timing of senescence pathways in petals.
Key words: Cysteine proteinases, etr1-1, flowers, KDEL-containing proteases, plants, petunias, SAG12, senescence
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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Senescence represents the last stage of flower development, ultimately culminating in the death of the petals. The senescence programme is regulated by co-ordinated changes in gene expression, and the later stages of senescence share many characteristics of programmed cell death (Rubinstein, 2000
; Jones, 2004). Flower petals provide an excellent model system for studies of senescence because they have a finite lifespan and their death is under tight developmental control. While flowers serve an essential role in sexual reproduction, the maintenance of petals is costly in terms of respiratory energy and water loss (Stead, 1992). The programmed senescence of the petals (collectively called the corolla), once a flower is pollinated or no longer receptive to pollination, allows the plant to break down macromolecules and organelles and remobilize their constituents to developing tissues. In support of this recycling programme, a number of genes encoding hydrolytic enzymes have been identified in screens for genes up-regulated during petal senescence (reviewed by Jones, 2004). Decreases in total nitrogen and phosphorus levels have also been detected in senescing petals (Nichols, 1976; Verlinden, 2003).
The degradation of proteins and the remobilization of amino acids to developing tissues is a prominent process during senescence (Solomon et al., 1999). Endopeptidases, or proteases, which degrade proteins by hydrolysing internal peptide bonds, are subsequently one of the most well-characterized cell death proteins in plants (Beers et al., 2000). These proteases are divided into the following subclasses based on their catalytic mechanisms: serine proteases (EC 3.4.21), cysteine proteases (EC 3.4.22), aspartic proteases (EC 3.4.23), metalloproteases (EC 3.4.24), and threonine proteases (EC 3.4.25). Experiments with class-specific inhibitors have attributed the protease activity associated with petal senescence primarily to the cysteine proteases (Stephenson and Rubinstein, 1998; Eason et al., 2002; Wagstaff et al., 2002). In support of their role in flower senescence, cysteine proteases up-regulated during petal senescence have been cloned from the petals of Dianthus caryophyllus (carnation) (Jones et al., 1995), Hemerocallis spp. (daylily) (Valpuesta et al., 1995; Guerrero et al., 1998), Alstroemeria peruviana (Wagstaff et al., 2002), Sandersonia aurantiaca (Eason et al., 2002), Narcissus pseudonarcissus (daffodil) (Hunter et al., 2002), and Gladiolus grandiflora (Arora and Singh, 2004).
Floral senescence in many species is regulated by the plant hormone ethylene (Woltering and van Doorn, 1988; Borochov and Woodson, 1989). In these flowers, petal senescence is associated with an increase in endogenous ethylene production, and inhibitors of ethylene biosynthesis and action delay senescence (Lawton et al., 1990; Woodson et al., 1992; Jones and Woodson, 1997). The identification of mutants with impaired ethylene production or perception has allowed for a more precise analysis of ethylene function during senescence than could be obtained from inhibitor studies alone (Chang et al., 1993). The Arabidopsis thaliana ethylene-insensitive mutant etr1-1 exhibits a delayed leaf senescence phenotype, and this delay is accompanied by a corresponding delay in the expression of senescence-associated genes (SAGs) (Grbic and Bleecker, 1995). Although SAG induction was delayed in etr1-1 plants, expression levels were similar to those detected in wild-type Arabidopsis leaves. This led to the conclusion that ethylene was affecting the timing of leaf senescence, but was not required for the execution of the senescence programme once it had begun. It has recently been reported that nuclear DNA fragmentation and the induction of a Co2+-dependent nuclease (PhNUC1) were delayed during corolla senescence in ethylene-insensitive, 35S:etr1-1 transgenic petunias (Langston et al., 2005). These studies suggest that ethylene is involved in modulating the timing of flower senescence, but it is still unclear whether there are components of the senescence pathways in petals that are dependent on ethylene.
Petuniaxhybrida has served as a model ethylene-sensitive plant for the molecular and biochemical analysis of flower senescence. Transformation of petunia with the etr1-1 gene from Arabidopsis confers ethylene insensitivity or significantly reduced sensitivity and delays flower senescence (Wilkinson et al., 1997; Gubrium et al., 2000; Shibuya et al., 2004). In this paper, 35S:etr1-1 transgenic petunias have been used to study how ethylene regulates flower senescence. To compare the senescence programmes in ethylene-sensitive (MD) and ethylene-insensitive (etr-44568) flowers, a comparative analysis was conducted of age-related changes in total protein, protease activity, and the expression of nine cysteine protease genes in the petals of MD and etr-44568 petunias. These characteristics were chosen for comparison because protein catabolism is a central component of the senescence programme in petals (Rubinstein, 2000; Jones, 2004). This is the first report of the differential regulation of individual cysteine protease genes by ethylene during flower development, and supports the idea that senescence-induced gene expression in petals occurs via ethylene-dependent and ethylene-independent signalling pathways.
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Materials and methods |
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Plant materials
Petuniaxhybrida cv. Mitchell Diploid (MD; wild type) and transgenic 35S:etr1-1 (etr-44568, Wilkinson et al., 1997
; Shibuya et al., 2004) plants were used in all experiments. All transgenic plants were homozygous for the transgene and were from the T5 generation. Petunias were grown under greenhouse conditions, with temperatures set at 22/18 °C day/night and a 13 h photoperiod supplemented by metal halide and high pressure sodium lights. Seeds were treated with 100 µl l–1 GA3 for 24 h and sown in cell packs on top of soil-less mix (Promix BX, Premier Horticulture, Quebec, Canada). All plants were established in the greenhouse after seed germination, and were moved to 15 cm plastic pots after 4 weeks. Plants were fertilized at each irrigation with 150 mg l–1 nitrogen from Peter's 15N-5P-15K (The Scotts Co., Marysville, OH). Flowers were emasculated 1 d before flowers were fully open to prevent self-pollination and were left on the flower to senesce naturally. Corollas were collected from MD petunias at 0, 2, 4, 6, and 8 d after flower opening (dao) and from etr-44568 petunias at 0, 2, 4, 6, 8, 10, 12, 14, and 16 dao. Styles (+stigmas) and ovaries were collected from MD flowers at 0 and 8 dao. MD flowers were pollinated by brushing pollen from freshly dehisced anthers onto the stigmas. Corollas, ovaries, and styles (+stigmas) were collected at 3 d after pollination when the flowers were wilted. Green, fully expanded leaves were collected from the top of the plant (green leaves) and naturally senescing leaves that were greater than 75% yellow were collected from the bottom (senescing leaves) of 24-week-old plants. Fully expanded green leaves were also collected 3 h after wounding with a wire brush (wounded leaves). Root and stem tissue was collected from 12-week-old plants and pollen was collected from flowers on the day of anther dehiscence. All tissues were frozen in liquid nitrogen and stored at –80 °C until used for protein extraction, protease activity, or RNA extraction. Fresh weights were measured immediately before freezing. At least six corollas, styles, or ovaries were pooled for each time point. All experiments were conducted at least three times with independently collected and extracted tissues unless otherwise noted.
Total protein determination
Frozen tissue was powdered in liquid nitrogen and ground in 2 ml extraction buffer (100 mM NaPO4, 2 mM EDTA, 20 mM DTT, 100 µM leupeptin, 5 µM pepstatin, 1 mM PMSF, 10 mM 1,10-phenanthroline, and 10 µM E-64) per corolla. Samples were centrifuged at 10 000 g at 4 °C for 15 min. The supernatant was used for protein quantification by the Bradford method (Bio-Rad Protein Assay Kit, Hercules, CA) using a BSA standard curve.
Protease activity
In vitro protease activity was determined using the synthetic substrate azocasein based on a method modified from Sarath et al. (1989). Corollas were powdered in liquid nitrogen and ground in ice-cold extraction buffer (2 ml per corolla) containing 100 mM NaPO4 (pH 6.2) and 20 mM DTT. The samples were transferred to centrifuge tubes and centrifuged at 10 000 g at 4 °C for 15 min. The supernatant was stored at –80 °C until used in activity assays. Protease activity of total protein extracts was determined by adding 150 µl protein extract to 250 µl 100 mM NaPO4 buffer (pH 6.2) containing 1% azocasein. Samples were incubated for 5 h at 37 °C. Optimum buffer composition, incubation duration and temperature, and assay pH for protease activity against azocasein were determined in preliminary experiments (data not shown). Reactions were terminated by adding 1.2 ml 10% TCA. Samples were mixed thoroughly and incubated at room temperature for 15 min. Samples were then clarified by centrifugation at 8000 g for 8 min and the supernatant was alkalinized with 1.4 ml 1.0 M NaOH. Blanks were prepared by incubating the protein and azocasein separately and adding the substrate to the sample after the addition of TCA. Absorbance was measured at 490 nm using a Beckman DU640 Spectrophotometer (Beckman Coulter, Fullerton, CA). Sample protease activity was defined in arbitrary units where 1 unit was equivalent to a change of 0.01 absorbance units h–1 at 490 nm.
Class-specific protease inhibitors were incorporated into the assay to determine the specificity of the protease activity detected in corolla total protein extracts. Individual protease inhibitors were incubated with the total protein extract for 30 min at room temperature prior to the addition of the azocasein substrate. Protease inhibitors and final concentrations used included: E-64 (L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane; 10 µM), leupeptin (100 µM), pepstatin (5 µM), 1,10-phenanthroline (10 mM), PMSF (phenylmethanesulphonyl fluoride; 1 mM) (Sigma, St Louis, MO). Blanks that included the specific protease inhibitors were prepared as described above.
Cysteine protease genes
Nine genes encoding putative cysteine proteases were identified from a search of the Petunia BlastQuest EST database developed at the University of Florida. These cysteine proteases were named PhCP2 to PhCP10 (Petuniaxhybrida Cysteine Protease) and sequences have been submitted to GenBank (Accession nos AY662988–AY662996). Full-length sequencing of the selected cDNA clones was conducted by Amplicon Express (Pullman, WA). Sequence analysis was performed with Sequencher V4.1 (Ann Arbor, MI) and software available from NCBI (http://www.ncbi.nlm.nih.gov/Genbank/).
RNA extraction, RT-PCR, and gel blot analysis
Total RNA from petunia corollas, ovaries, styles, pollen, leaves, stems, and roots was isolated using Trizol (GibcoBRL, Rockville, MA). Two micrograms of total RNA was reverse transcribed at 37 °C for 2 h using Omniscript reverse transcriptase (Qiagen, Valencia, California). Two microlitres of cDNA was used as template for PCR amplication using Master Taq (Eppendorf, Hamburg, Germany) and specific primers to the nine cysteine protease genes (Table 1). Actin was amplified as a control. Actin primers were constructed to the tomato TOM51 actin cDNA (GenBank accession no. U60481) and included F 5'-GTGTTGGACTCTGGTGATGG-3' and R 5'-TCAGCAGTGGTGGTGAACAT-3'. PCR was conducted for 26 cycles of 94 °C for 2 min, 60 °C for 2 min, and 72 °C for 2 min using a Mastercycler gradient thermocycler (Eppendorf, Hamburg, Germany). PCR products were separated on 1.0% agarose gels and stained with ethidium bromide. Negative controls had no template DNA and positive controls included the cysteine protease or actin cDNAs in pBluescipt (Stratagene, LaJolla, CA).
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Fifteen micrograms of total RNA was size separated through a denaturing 1.2% agarose gel containing formaldehyde, and subsequently, transferred overnight onto Hybond N membrane (Amersham Pharmacia Biotech, Piscataway, NJ) by capillary transfer in 10x SSC. The RNA was cross-linked using a UV crosslinker at 50 mV cm–2 (Bio-Rad Laboratories, Hercules, CA). Radiolabelled, cDNA probes were generated by PCR using gene-specific primers (Table 1) and
32P-CTP (PerkinElmer, Boston, MA). Prehybidization and hybidizations were performed as previously described (Jones et al., 1995). Hybridization signals were visualized using a Storm 860 PhosphoImager and quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Transcript levels were normalized to rRNA levels to correct for any differences in RNA loading.
Western blot analysis
Frozen tissue was powdered in liquid nitrogen, ground in extraction buffer [100 mM TRIS–HCl, pH 7.6, 10 mM MgSO4, 10 mM DTT, complete mini protease inhibitor cocktail tablet (1 tablet per 10 ml; Roche Diagnostics Corporation, Indianapolis, IN)], and incubated on ice for 1 h with periodic vortexing. Samples were centrifuged at 20 000 g at 4 °C for 10 min. Protein in the supernatant was quantified by the Bradford method (Bio-Rad Protein Assay Kit, Hercules, CA) using a BSA standard curve. Equivalent amounts of protein (10 µg) were size-fractionated by SDS–PAGE on 10% acrylamide gels. After electroblotting on to Immun-Blot7 PVDF membrane (Bio-Rad, Hercules, CA), blots were blocked with 1% BSA in 50 mM TRIS–HCl, 150 mM NaCl, 0.3% Tween 20 (v/v), pH 8, overnight at room temperature. Blots were then incubated with primary antibody raised against the purified CysEP (diluted 1:1000 in blocking solutions, Schmid et al., 1998) for 2 h, and subsequently washed with TRIS-buffered saline (TBS) containing 0.5% (v/v) Tween-20 for 5x5 min each. Immunoreactive proteins were visualized using the secondary antibody alkaline phosphatase-coupled sheep anti-rabbit IgG with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate, toluidine salt (Roche Diagnostics Corporation, Indianapolis, IN) as a substrate.
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Results |
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Corolla wilting and fresh weight declines are delayed by approximately 8 d in etr-44568 transgenic petunias
MD petunias exhibited the first visible symptoms of senescence, wilting of the corolla, at approximately 8 d after flower opening (dao) (8.4±0.26 d, n=18; Fig. 1A). Flower senescence in etr-44568 flowers was significantly delayed, but more variability was observed in the number of days to corolla wilting. The average time to wilting was 15.6±0.97 d, (n=18) with some flowers wilted on day 14 and some as late as day 18. Under these growing conditions the fresh weight of etr-44568 flowers was slightly less than that of MD on the day of flower opening (0 dao) (Fig. 1B). At 2 dao the fresh weight of both MD and etr-44568 flowers increased compared with 0 dao. Corolla fresh weights of both genotypes remained constant from 4–6 dao. By day 8, the fresh weight of MD corollas had decreased to approximately 84% of the corollas mean fresh weight at 0 d, and this decrease coincided with corolla wilting. The fresh weight of etr-44568 corollas remained constant until 14–16 dao, which also coincided with wilting of the corolla.
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Comparative changes in total protein content and protease activity in the corollas of MD and etr-44568 petunias
On the day of flower opening, etr-44568 flowers had less total protein per corolla than MD flowers (Fig. 2A). The level of soluble proteins in MD corollas remained relatively constant from 0 to 4 dao and then began to decrease. The protein content of wilted corollas at 8 dao was only 46% of that measured from corollas at 0 d. Protein levels increased slightly in etr-44568 corollas at 2 dao and remained relatively constant until 10 dao. By 16 dao, protein levels in etr-44568 corollas had decreased to levels that were comparable with those measured from senescent MD corollas at 8 dao.
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In vitro protease activity was determined against the synthetic protease substrate azocasein (Fig. 2B). Low levels of protease activity were detected in total protein extracts from MD and etr-44568 corollas on the day of flower opening. Protease activity increased at 2 dao, and a greater than 2-fold increase in activity was subsequently measured from day 2 to day 4 in both MD and etr-44568 corollas. Smaller increases in activity were measured between 4 and 6 dao in both genotypes. Protease activity was highest in MD corollas late in the senescence programme when petals were wilted (8 dao). Protease activity increased more gradually during the later part of etr-44568 corolla senescence, and maximum protease activity was detected at 16 dao when petals were wilted. The maximum levels of protease activity measured in MD petals at 8 dao were similar to those measured in etr-44568 petals at 16 dao.
Class-specific protease inhibitors were incorporated into the in vitro protease activity assays to determine which classes of proteases were responsible for the increases in protease activity detected during the senescence of MD and etr-44568 corollas (Fig. 3). At 8 dao, when MD corollas had the highest activity, the greatest inhibition of protease activity was measured in total protein extracts incubated with E-64 and leupeptin. These samples had only 9% and 11%, respectively, of the activity detected in control extracts with no inhibitors. E-64 is an irreversible inhibitor of cysteine proteases, and leupeptin inhibits trypsin-like serine and most cysteine proteases. E-64 and leupeptin also inhibited protease activity in etr-44568 corolla extracts at 8 dao and 16 dao. In vitro protease activity in 16 d etr-44568 corollas was only 11% and 7% of control activity when treated with E-64 and leupeptin, respectively. The aspartic protease inhibitor, pepstatin, had no effect on protease activity in either MD or etr-44568 corollas. 1,10-phenanthroline, a metalloprotease inhibitor, resulted in a slight reduction in activity in both genotypes. PMSF, which inhibits serine proteases and some cysteine proteases, had no significant effect on activity in either MD or etr-44568 corollas.
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Differential regulation of cysteine protease genes during the senescence of MD and etr-44568 petunias
Inhibitor studies indicated that the majority of the protease activity during senescence was the result of proteases in the cysteine protease class. Therefore, putative cysteine protease genes were identified by searching ESTs generated by random sequencing of several petunia flower cDNA libraries. This search initially identified nine unique ESTs with homology to known cysteine proteases (CP). These clones were named PhCP2–PhCP10 (Petuniaxhybrida Cysteine Protease), and represent cDNAs ranging in size from 629 bp to 1041 bp. (Table 1). PhCP1 was reserved for a previously identified petunia CP (PeTh3, GenBank no. U31094; Tournaire et al., 1996
). The PhCPs have the highest homology to members of the papain-like cysteine proteases (family C1A), and an NCBI conserved domain search identified putative Peptidase C1 domains in the predicted amino acid sequences of all nine PhCPs.
The predicted amino acid sequences of the petunia cysteine proteases were aligned with the most homologous plant proteases, including those identified from flowers, using ClustalW. The phylogenetic relationship of these proteases is shown in Fig. 4. PhCP2 groups with PhCP8 and PhCP9 and other papain-type cysteine proteases that are up-regulated during senescence and by stresses including temperature, drought, and Phytophthora infestans infection (Drake et al., 1996; Avrova et al., 1999). PhCP3 groups with a cluster of CPs up-regulated by drought, circadian rhythm, and wounding (Linthorst et al., 1993). It is most homologous to a CP from Solanum melongena (egg plant) that is up-regulated during developmental events associated with programmed cell death (Xu and Chye, 1999). PhCP4 is most homologous to cathepsin B-like cysteine proteases from Solanum tuberosum (potato) (Avrova et al., 2004) and Nicotiana rustica (Aztec tobacco) (Lidgett et al., 1995). These genes are up-regulated following Phytophthora infestans infection and wounding, respectively. PhCP5 is most similar to CP isoform III from Ipomea batatas (sweet potato). It also groups with a number of CPs identified from the tepals of ethylene-insensitive flowers, including PRT22 from Sandersonia, IhCYS3 from Irisxhollandica, and DAFSAF2 from daffodil (Eason et al., 2002; Hunter et al., 2002). The predicted polypeptide of PhCP6 contains a C-terminal KDEL motif, and sequence alignments group it with other KDEL-containing cysteine proteases. This motif is thought to function as an endoplasmic reticulum retention signal for soluble proteins (Schmid et al., 1999). PhCP6 is most similar to Cys-EP, the Ricinus communis (castor bean) cysteine protease involved in programmed cell death of the endosperm. PhCP7 has the highest homology to a CP from Trifolium repens (white clover) and a xylem endopeptidase from Arabidopsis involved in PCD during tracheary element differentiation (Zhao et al., 2000; Asp et al., 2004). PhCP10 groups with a number of CP genes that have been reported to have senescence-specific patterns of transcript accumulation, including SAG12 from Arabidopsis, and SAG12 homologues from Brassica napus (rape) (BnSAG12-2) and Nicotiana tabacum (tobacco) (NtCP1) (Weaver et al., 1998; Noh and Amasino, 1999).
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RT-PCR experiments using gene-specific primers for all PhCPs were conducted to determine where in the plant the PhCP transcripts were detected and to determine if they were detectable in non-senescing (0) and senescing (3P and 8U) flower tissues (Fig. 5). Naturally senescing, unpollinated MD flowers were collected at 8 dao (8U), and pollinated MD flowers were collected at 3 d after pollination (3P) when they were senescent (corollas were wilted). All nine PhCPs were detected in flower corollas. Only PhCP10 was detected in senescing but not in non-senescing corollas. PhCP10 was also detected in senescing, but not in non-senescing, green leaves. Transcripts for PhCP2, PhCP3, PhCP4, PhCP5, PhCP7, and PhCP9 were detected in flowers, leaves, stems, and roots. PhCP6 transcripts were most abundant in flower tissues, and barely detectable in green leaves and stems. PhCP8 was not detectable in pollen or roots. While PhCP8 transcripts were detected in ovaries from flowers on the day of flower opening (0), they were not detected in ovaries from naturally senescing (8U) or pollinated flowers (3P).
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Northern blot analysis was conducted to quantify changes in transcript abundance during corolla senescence, and to identify those cysteine proteases involved in petal senescence (Fig. 6). Some of the PhCPs had the highest transcript accumulation in senescing corollas, while some were down-regulated during flower development. In MD corollas PhCP2, PhCP3, PhCP5, PhCP8, PhCP9, and PhCP10 showed patterns of up- regulation during senescence. These were referred to as senescence-associated cysteine proteases or SACPs. PhCP8 and PhCP10 had maximum transcript abundance at 6 dao, just before the fresh weight decline and visible symptoms of corolla wilting, while maximum transcript abundance of PhCP2, PhCP3, PhCP5, and PhCP9 was detected at 8 dao, when corollas were wilted and protease activity was highest. Transcripts for all of the SACPs were detected in non-senescing corollas from 0 to 4 dao, except for PhCP10, which was specific to senescing corollas. PhCP5, which had relatively high constitutive levels of transcript at 0–6 dao, had the smallest up-regulation at the later stages of senescence when compared with the other SACP genes.
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In etr-44568 corollas, maximum transcript abundance of the SACPs, PhCP2, PhCP8, PhCP9, and PhCP10 was delayed to 14–16 dao and corresponded with corolla wilting. By contrast, maximum transcript abundance of PhCP3 was detected at 8 dao and had returned to lower, basal levels by 14–16 dao. Relative mRNA abundance of PhCP5 was also similar in WT and etr-44568 corollas at 8 dao, but mRNA levels continued to increase from 8–14 dao in the etr-44568 corollas.
PhCP4, PhCP6, and PhCP7 were down-regulated during the development of WT and etr-44568 corollas. In etr-44568, PhCP7 gradually decreased by 16 dao to levels detected in comparably wilted MD flowers at 8 dao. PhCP4 and PhCP6 transcript abundance decreased more rapidly than PhCP7, with large decreases detected at 2 dao and 4 dao in etr-44568 and MD corollas, respectively.
Western blot analysis of CysEP-like proteases
In contrast to the KDEL-containing CysEP-like proteases that have been identified in other flowers, PhCP6 was found to be transcriptionally down-regulated during petunia petal senescence (Fig. 5; Guerrero et al., 1998; Eason et al., 2002). Western blot analysis of protein extracts from naturally senescing (0, 2, 4, 6, and 8 dao) petunia corollas confirmed that a CysEP-like protein decreased in abundance as MD corollas senesced. The antibody raised against CysEP (Schmid et al., 1998) cross-reacted with one protein band from petunia corollas (c. 37.5 kDa; Fig. 7). The abundance of this 37.5 kDa protein declined from 0 dao to 8 dao.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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A common feature of programmed cell death during organ senescence is the degradation of proteins (Huffaker, 1990
). Petal senescence has been associated with decreased protein levels, increased proteolytic activity, and the up-regulation of cysteine protease genes (reviewed in Jones, 2004). While one of the earliest reports of a cysteine protease involved in petal senescence was from ethylene-sensitive carnation flowers (Jones et al., 1995), most studies of proteases and petal senescence have utilized species in which petal senescence is not regulated by ethylene (i.e. ethylene-insensitive flowers) (Guerrero et al., 1998; Stephenson and Rubinstein, 1998; Eason et al., 2002; Hunter et al., 2002; Wagstaff et al., 2002; Arora and Singh, 2004; Pak and van Doorn, 2005). Several senescence-associated cysteine protease genes have been reported to increase in abundance following ethylene treatment (Cervantes et al., 1994; Alonso and Granell, 1995; Jones et al., 1995; Weaver et al., 1998; Cercos et al., 1999; Chen et al., 2002), but the role of ethylene in the initiation and execution of protein catabolism during petal senescence is not clear. A comparative study of age-related changes in total protein, protease activity, and cysteine protease gene expression in corollas from the ethylene-sensitive flowers of Petuniaxhybrida cv. Mitchell Diploid (MD) and the ethylene-insensitive flowers of transgenic 35S:etr1-1 petunias (etr-44568) has therefore been conducted.
Transformation of petunias with the mutated ethylene receptor gene (etr1-1) from Arabidopsis reduces ethylene sensitivity in flowers and delays senescence (Wilkinson et al., 1997; Gubrium et al., 2000; Shibuya et al., 2004). Multiple independent transgenic lines have been generated with varying degrees of ethylene sensitivity. To determine the level of ethylene sensitivity of 35S:etr1-1 lines at the molecular level, the induction of PhEIL1, a transcription factor involved in ethylene signal transduction, was determined following ethylene treatment of flowers (Shibuya et al., 2004). In MD corollas, expression of PhEIL1 was induced 370% by exogenous ethylene compared with untreated controls. PhEIL1 expression was not induced in ethylene-treated etr-44568 flowers, indicating that these flowers are insensitive to ethylene or, at the very least, have greatly reduced ethylene sensitivity. By contrast, in etr-56, a transgenic line characterized as having reduced ethylene sensitivity, PhEIL1 was induced 187% by ethylene.
Corolla senescence in both ethylene-sensitive MD and ethylene-insensitive etr-44568 petunias was accompanied by increased proteolytic activity and a decline in total protein levels. Quantitatively, the increase in proteolytic activity and the decrease in protein content were similar in etr-44568 and MD corollas, suggesting that the execution of protein catabolism during senescence is not dependent on ethylene signalling. On the other hand, the temporal regulation of protease activity and protein degradation differed between MD and etr-44568 flowers. Corolla senescence was delayed by approximately 8 d in etr-44569 flowers. Maximum protease activity and net protein losses were similarly delayed and corresponded with the wilting of the etr-44568 corollas. It was recently reported that DNA fragmentation and the induction of a senescence-specific nuclease (PhNUC1) were also delayed in 35S:etr1-1 transgenic petunias (Langston et al., 2005). Similarly, Burdon and Sexton (1993) reported that ethylene accelerates and co-ordinates petal abscission and senescence in raspberry flowers, as both processes will eventually occur even in the absence of ethylene. These studies support the role of ethylene as a modulator of senescence timing in petals.
While the senescence-related induction of proteolytic activity appeared to be merely delayed in etr-44568 corollas, it was not known whether the same proteases or even the same class of proteases was responsible for the proteolytic activity measured in MD and etr-44568 corollas. It was found that the protease activity detected in protein extracts from both senescing MD and etr-44568 corollas was mainly due to cysteine proteases (
90%), and to a much lesser extent metalloproteases (<10%) (Fig. 3). In similar inhibitor studies with the ethylene-insensitive flowers, Sandersonia and Iris, approximately 50% of the protease activity measured in senescing tepals was attributed to cysteine proteases (Eason et al., 2002; Pak and van Doorn, 2005). Treatment of Sandersonia and Iris flowers with the cysteine protease inhibitors, leupeptin and E-64, respectively, was also found to delay visible symptoms of senescence and decrease endogenous protease activity (Eason et al., 2002; Pak and van Doorn, 2005). An increase in cysteine protease activity would appear to be a common feature of petal senescence in both ethylene-sensitive and ethylene-insensitive flowers.
In support of the role of cysteine proteases in petunia flower senescence, it was possible to identify nine cysteine protease genes that were expressed in corollas. Three of the nine cysteine protease genes (PhCP4, PhCP6, PhCP7) were down-regulated during flower ageing in both MD and etr-44568 (Fig. 6). Six cysteine protease genes (PhCP2, PhCP3, PhCP5, PhCP8, PhCP9, PhCP10) were up-regulated during the natural ageing of corollas, but the temporal expression patterns of some of these genes differed in MD and etr-44568 corollas.
The pattern of decreasing mRNA abundance of PhCP4, PhCP6, and PhCP7 during flower ageing suggests that this group of cysteine proteases is most likely involved in the regulation of general protein turnover and cellular maintenance during the growth and development of the petals. Ethylene would not appear to be involved in co-ordinating the expression of these genes, as the patterns of down-regulation following flower opening were similar in MD and etr-44568 corollas. Of particular interest within this group of genes is PhCP6, which has high homology to CysEP from castor bean and other KDEL-containing cysteine proteases (Fig. 4). CysEP is localized within membrane-bound organelles called ricinosomes that are formed at the beginning of PCD (Schmid et al., 1998). Acidification of the ricinosomes during the later stages of cell death causes activation and release of the CysEP following cleavage of the N-terminal pro-peptide and the C-terminal KDEL (Schmid et al., 2001). Ricinosomes were originally identified in the endosperm of castor beans, but have also been detected in senescing daylily petals (Schmid et al., 1999). In contrast to PhCP6, transcript abundance of the KDEL-containing cysteine proteases from Sandersonia (PRT5) and daylily (SEN102 and SEN11) increased during tepal senescence (Guerrero et al., 1998; Eason et al., 2002). Western analysis using an antibody raised against the active form of CysEP (Schmid et al., 1998) recognized a protein of approximately 37.5 kDa in petunias that also decreased in abundance during corolla senescence. These experiments suggest that the CysEP-like proteases in petunia corollas are not likely to be involved in the large-scale proteolysis that accompanies the later stages of petal senescence, but that they may be involved in processes early in flower development that involve PCD.
The majority of the cysteine protease genes from petunia exhibited senescence-associated (SA) increases in transcript abundance (SACPs; PhCP2, PhCP3, PhCP5, PhCP8, PhCP9, and PhCP10) in MD corollas. All SACPs, except for PhCP10, were detected at basal levels in non-senescing tissues and increased in abundance during senescence. The expression of these SACPs suggests that their major role during senescence is catalysing the large-scale degradation of proteins that accompanies petal wilting, but that they may also have a role in protein turnover throughout flower development. The majority of the senescence-associated genes (SAGs) that have been identified from leaves and petals show a similar expression profile, and only a few SAGs have been detected only in senescing tissues (Buchanan-Wollaston, 1997; Jones, 2004).
PhCP10 was the only petunia SACP that had senescence-specific expression. Transcripts were not detectable in non-senescing floral or vegetative tissues, but were induced during the senescence of petals, styles, and leaves. PhCP10 has high homology to SAG12 from Arabidopsis and the SAG12 homologues from tobacco and B. napus (Fig. 4). SAG12 has no detectable expression in young leaves and is induced in older leaves during age-mediated senescence. Of the Arabidopsis senescence-associated genes, SAG12 is suggested to be one of the best molecular markers of leaf senescence (Weaver et al., 1998). Similarly, the SAG12 homologue, PhCP10, would appear to be an excellent molecular marker for petal senescence.
It has been suggested by many researchers that senescence-associated proteases are functioning in the complete proteolysis that accompanies senescence and programmed cell death and allows for nutrient remobilization to sink tissues (Rubinstein, 2000; Jones, 2004). There is increasing evidence that proteases may have a regulatory role in plant growth and development by selectively cleaving and subsequently activating enzymes, including other proteases (Beers et al., 2004). In this way, their function during senescence may be similar to the initiator caspases functioning in animal PCD pathways (Woltering et al., 2002). PhCP10 and PhCP8 had maximum expression at 6 dao, before the visible symptoms of senescence, with decreasing abundance at 8 dao when the other SACPs had maximum transcript abundance. These patterns suggest that PhCP10 and PhCP8 may function earlier in the senescence programme, and they may be involved in initiating signalling pathways by cleaving other senescence-associated proteins. Three cysteine proteases that are up-regulated during tepal senescence have been reported in Sandersonia flowers (Eason et al., 2002). One of these CPs, PRT5, was reported to be specifically expressed during flower senescence, and was not detected in immature flowers or vegetative tissues. Like PhCP10, PRT5 mRNAs began to accumulate earlier than the other senescence-associated CPs and before visible symptoms of senescence were apparent (Eason et al., 2002).
A subgroup of the petunia SACPs (PhCP2, PhCP8, PhCP9, and PhCP10) also had senescence-associated increases in transcript abundance in etr-44568 corollas at 14–16 dao when these flowers were wilting. Although expression was delayed, relative transcript abundance was similar to that measured in senescing MD corollas. Delays in leaf senescence in the Arabidopsis etr1-1 mutant were also accompanied by corresponding delays in the expression of senescence-associated genes (Grbic and Bleecker, 1995). PhCP2, PhCP8, and PhCP9 have high homology to the tomato cysteine protease, C14 (SenU2) (Fig. 4). This cysteine protease was originally detected in tomato fruits following low temperature stress (Schaffer and Fischer, 1988), but it is also involved in leaf senescence (Drake et al., 1996). C14 (SenU2) transcripts were detected in young fully expanded leaves, increased in abundance during ageing, and reached maximum abundance at the later stages of leaf senescence (Drake et al., 1996). In transgenic tomato plants deficient in ethylene biosynthesis, visible symptoms of leaf senescence and the enhanced accumulation of SenU2 (C14) mRNAs were delayed (Drake et al., 1996). This is very similar to the expression profile of PhCP2, PhCP8, and PhCP9 during corolla development in MD and etr-44568 petunias, and it suggests that ethylene is similarly regulating the timing of proteolysis during the senescence of leaves and petals.
In contrast to the expression of PhCP2, PhCP8, PhCP9, and PhCP10, transcript accumulation of PhCP3 and PhCP5 was not delayed in etr-44568 corollas. The temporal expression profile of PhCP3 was similar in etr-44568 and MD corollas. PhCP3 reached maximum transcript abundance at 8 dao, and declined by 14–16 dao when the etr-44568 corollas were wilting. Relative abundance of PhCP5 transcripts was similar in MD and etr-44568 corollas at 8 dao, but continued gradually to increase until 14 dao. These expression profiles suggest that PhCP3 and PhCP5 may encode the cysteine proteases responsible for the enhanced activity detected in etr-44568 corollas at 8 dao. Phylogenetic analysis (Fig. 4) shows that PhCP3 and PhCP5 have high homology to CPs from the ethylene-insensitive flowers Iris, Sandersonia, and daffodil. Both DAFSAG2 and PRT22, from daffodil and Sandersonia, respectively, were detected at low levels in young flowers and increased in abundance during the later stages of senescence (Eason et al., 2002; Hunter et al., 2002). PhCP3 and PhCP5 appear to be regulated during flower development independent of ethylene action, and may be regulated by age-related factors similar to those controlling senescence-associated expression of cysteine proteases in ethylene-insensitive, non-climacteric flowers. Gene expression studies using etr1-1 Arabidopsis mutants led to similar conclusions regarding ethylene's regulation of abscission. While abscission could be accelerated by ethylene, it was not required for the normal abscission programme. These studies also identified both ethylene-dependent and ethylene-independent components of the abscission process in Arabidopsis (Patterson and Bleecker, 2004).
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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It has been shown that ethylene sensitivity regulates the timing of flower senescence in part by delaying the increased proteolytic activity and subsequent protein degradation that are an integral component of the senescence programme in petunia corollas. Expression profiles of the cysteine proteases during flower development and senescence identified both genes that were regulated by ethylene and those that were independent of ethylene. Expression of most of the senescence-associated cysteine protease genes was delayed but not prevented in etr1-1 flowers, and these delays corresponded with corolla wilting. The delay in the progression of senescence in ethylene-insensitive flowers is consistent with the concept that ethylene acts as a modulator of senescence pathways.
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Acknowledgements |
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This research was funded by the American Floral Endowment, the Floriculture and Nursery Research Initiative and the Floriculture Industry Research and Scholarship Trust (FIRST) Gus Poesch Endowment. Salaries and research support were provided, in part, by State and Federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University (Journal article number HCS 05-13). We acknowledge Dr Tea Meulia at the OSU Molecular and Cellular Imaging Center for use of the PhosphoImager and GeneQuant Software, and we thank Sarah Negley for excellent technical assistance in the laboratory and the greenhouse.
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Footnotes |
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* Cysteine protease sequences in this paper include GenBank Accession numbers AY662988–AY662996.
Abbreviations: CP, cysteine protease; dao, days after opening; E-64, L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane; EST, expression sequence tag; MD, Mitchell Diploid; PCD, programmed cell death; PMSF, phenylmethanesulphonyl fluoride; SA, senescence-associated; SAG, senescence-associated gene.
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References |
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