JXB Advance Access originally published online on July 13, 2007
Journal of Experimental Botany 2007 58(11):2873-2885; doi:10.1093/jxb/erm076
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RESEARCH PAPER |
Sucrose prevents up-regulation of senescence-associated genes in carnation petals
1Agrotechnology and Food Science, Wageningen University and Research Centre, PO Box 17, 6700 AA Wageningen, The Netherlands
2Plant Research International, Wageningen University and Research Centre, PO Box 16, 6700 AA Wageningen, The Netherlands
To whom correspondence should be addressed. E-mail: wouter.vandoorn{at}wur.nl
Received 24 August 2006; Revised 2 March 2007 Accepted 5 March 2007
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Abstract |
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cDNA microarrays were used to characterize senescence-associated gene expression in petals of cut carnation (Dianthus caryophyllus) flowers, sampled from anthesis to the first senescence symptoms. The population of PCR fragments spotted on these microarrays was enriched for flower-specific and senescence-specific genes, using subtractive hybridization. About 90% of the transcripts showed a large increase in quantity, approximately 25% transiently, and about 65% throughout the 7 d experiment. Treatment with silver thiosulphate (STS), which blocks the ethylene receptor and prevented the normal senescence symptoms, prevented the up-regulation of almost all of these genes. Sucrose treatment also considerably delayed visible senescence. Its effect on gene expression was very similar to that of STS, suggesting that soluble sugars act as a repressor of ethylene signal transduction. Two fragments that encoded a carnation EIN3-like (EIL) protein were isolated, some of which are key transcription factors that control ethylene response genes. One of these (Dc-EIL3) was up-regulated during senescence. Its up-regulation was delayed by STS and prevented by sucrose. Sucrose, therefore, seems to repress ethylene signalling, in part, by preventing up-regulation of Dc-EIL3. Some other transcription factors displayed an early increase in transcript abundance: a MYB-like DNA binding protein, a MYC protein, a MADS-box factor, and a zinc finger protein. Genes suggesting a role in senescence of hormones other than ethylene encoded an Aux/IAA protein, which regulate transcription of auxin-induced genes, and a cytokinin oxidase/dehydrogenase, which degrades cytokinin. Taken together, the results suggest a master switch during senescence, controlling the co-ordinated up-regulation of numerous ethylene response genes. Dc-EIL3 might be (part of) this master switch.
Key words: Carnation, Dianthus caryophyllus, ethylene, gene expression, microarray, petal, programmed cell death, senescence, sucrose
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Introduction |
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Senescence of leaves and petals is a highly regulated process that includes extensive breakdown of carbohydrates, proteins, lipids, and nucleic acids, prior to cellular death. In some species, such as carnation, flower senescence is regulated by endogenous ethylene. Carnation, therefore, often serves as a model for ethylene-sensitive flower senescence.
An autocatalytic rise in ethylene production precedes the senescence symptoms in carnation flowers. It is associated with increased transcription of genes encoding enzymes involved in ethylene biosynthesis, such as ACC synthase and ACC oxidase (Park et al., 1992). Genes that were up-regulated at about the same time included a cysteine protease (Jones et al., 1995), a glutathione S-transferase, (SR8; Meyer et al., 1991), a S-adenosylmethionine (SAM) synthase (Woodson et al., 1992), a putative β-glucosidase (SR5; Woodson, 1994), and a β-galactosidase (SR12; Raghothama et al., 1991). The expression of several of these genes was also induced after application of exogenous ethylene, supporting the view that their up-regulation during senescence is regulated by ethylene.
The initiation of autocatalytic ethylene production and the associated increase in gene expression might be the result of an increase in the sensitivity of flowers to its continuous low endogenous ethylene levels (Woodson and Lawton, 1988). If so, it is unclear how this increase in sensitivity comes about. It might be due to a decrease in cytokinin levels or to a decrease in soluble carbohydrate levels (van Doorn, 2004).
Some treatments are known to postpone or hasten the initiation of visible senescence in carnation flowers. Exposure to exogenous ethylene hastens senescence, whereas exposure to inhibitors of ethylene responses such as silver thiosulphate (STS) prevents the normal senescence symptoms. Sugar-treatment also markedly delayed visible senescence, associated with a delay in the rise of ethylene production. Exogenous ethylene had less effect in flowers that had been treated with sugars (Nichols, 1973; Mayak and Dilley, 1976), but the underlying molecular mechanism has remained elusive.
Several ethylene receptors are now known, and part of the ethylene signalling pathway has become elucidated. Downstream of the signalling pathway are EIN3 or EIN3-like (EIL) proteins, key transcription factors. In the absence of adequate ethylene levels these proteins become degraded by the 26S proteasome. In the presence of adequate ethylene, proteasome degradation is inhibited. EIN3 or EIL then reach adequate levels and attach to promoters, thereby allowing the expression of ethylene response genes (Guo and Ecker, 2004; Bischopp et al., 2006). In Arabidopsis, glucose was shown to decrease ethylene signalling by enhancing the proteasome-mediated degradation of EIN3 (Yanagisawa et al., 2003). In addition, glucose was found to down-regulate the expression of Arabidopsis EIN3 and EIL1 (Price et al., 2004). Three EIN3-like proteins have been identified in carnation petals (Iordachescu and Verlinden, 2005).
It was hypothesized that sucrose, known to delay the time to senescence in carnation petals, regulates the expression of a set of genes similar to those that are affected during senescence. To test this hypothesis, cDNA microarrays consisting of over 2000 cDNA transcripts derived from carnation petal tissue were made. In order to enrich for flower-specific and senescence-specific cDNAs, a subtraction was performed using tissue of the stem just beneath the flower head. The microarrays were used to analyse expression profiles during senescence of untreated, ethylene-treated, STS-treated, and sucrose-treated flowers. The results demonstrate up-regulation of a large number of genes during carnation petal senescence. Ethylene treatment advanced, and STS repressed the up-regulation of most of these genes. Similarly, sucrose treatment repressed the up-regulation of most of these genes, suggesting that sucrose might act as a repressor of ethylene action.
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Materials and methods |
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Plant material
Greenhouse-grown carnation flowers (Dianthus caryophyllus L. cv. Dover) were obtained from a local commercial grower. Flowers were harvested at the commercial harvesting stage (paintbrush stage). Flowers were placed in water directly after harvest and transported to the laboratory in water. In the laboratory, stems were recut to 50 cm length and flowers were placed in vases containing tap water.
Experiments were carried out in a climate-controlled room at 20 °C and 60% relative humidity, 15 µmol m–2 s–1 white light from fluorescent tubes for 12 h d–1, and 12 h darkness. Two discontinuous treatments started the first night after arrival of the flowers. Ethylene was applied at 200 nl l–1 for 18 h in closed 70 l stainless steel containers, the flowers remaining in water at all times. Silver thiosulphate (STS) was applied at 0.2 mM for 18 h. Flowers were subsequently placed in water. The sucrose treatment was continuous. Sucrose was applied in vase water at 25 g l–1, together with the antimicrobial compound sodium dichloroisocyanuric acid (DICA) at 150 mg l–1.
Petal tissue was sampled at intervals, always between 14.00 h and 16.00 h, immediately frozen in liquid nitrogen, homogenized to a fine powder and stored at –80 °C until further use. The time to visible senescence was determined in a parallel experiment using 10 flowers per treatment, all placed individually in vases. These flowers were examined once daily and were considered wilted when the petals had visibly lost turgor.
cDNA library construction, sequencing, BLAST analysis, and contig analysis
All molecular methods were the same as described previously by van Doorn et al. (2003). Total RNA was isolated according to Chang et al. (1993). 75 µg of RNA was used to isolate mRNA, using the Dynabeads mRNA Purification Kit (Dynal). After mRNA isolation, equal amounts from each developmental stage were combined. In order to enrich for flower-specific and senescence-specific cDNAs, a subtraction was performed using stem tissue (located just under the flower head) and the Clontech PCR-Select cDNA Subtraction Kit (with stem tissue as the driver), according to the manufacturer's instructions. The pGEM-T Easy Vector System (Promega) was used to construct a cDNA library from the resulting transcripts.
DNA sequencing was performed by Baseclear (Leiden, The Netherlands). DNA sequence homology searches were performed using the BLASTX algorithm (Altschul et al., 1997) against the nr (non-redundant) peptide sequence database (January 2006); hits with an E-value >1x10–8 were categorized as ‘no homology’. The Blastclust program was used to assemble transcripts to clusters (contigs) based on their (partial) nucleotide sequence. Contigs are formed by single-linkage clustering based on pairwise matches found using the Mega BLAST algorithm. Settings used: more than 95% identity (–S 95) and 0.5 length covering (–L 0.5) in one sequence of a pair (–b F).
Microarray preparation
2224 carnation DNA transcripts for arraying were obtained by PCR amplification on isolated plasmid DNA or directly on lysed bacteria. Plasmids were prepared from independent colonies of the plated cDNA library using Qiaprep Turbo miniprep kits (Qiagen). M13 universal and M13 reverse primers (Eurogentec) were used to amplify the insert in a 50 µl PCR reaction, containing 40 pmol of each primer, 1x PCR buffer (AP Biotech), 0.2 mM dNTPs, 1 unit Taq polymerase (AP Biotech), and either 10 ng template DNA or 10 µl of a x20 diluted overnight culture. Following a denaturation step at 94 °C for 30 s, 34 cycles were performed (10 s at 94 °C, 20 s at 55 °C, and 40 s at 72 °C). The amplification products were then purified using Qiaquick PCR purification kits (Qiagen) using 100 µl 1 mM TRIS (pH 8.0) as elution buffer. Eluates were dried to completion in a flow cabinet and dissolved in 10 µl 5x SSC, giving a final DNA concentration of 0.5–1.0 µg µl–1. In addition, PCR transcripts derived from the following sources were included on the microarray: (i) three non-plant genes as negative controls, used for the estimation of background signal: yeast aspartate kinase (Genbank accession number J03526), imidazoleglycerolphosphate dehydratase (accession number Z75110), phosphoribosylaminoimidazole carboxylase (Z75036) (in 8-fold); (ii) the complete coding sequence of the firefly luciferase gene (in 24-fold), and three partial luciferase transcripts encompassing the 5'-, middle- and 3'-part of the gene (in 8-fold). As the samples were spiked with luciferase mRNA prior to labelling this allowed for correction of the expression ratios for channel-specific effects. The partial luciferase transcripts were also used to monitor the integrity of the labelled sample cDNA.
Microarrays were spotted on GAPS amino silane-coated glass slides (Corning) using a PixSys 7500 arrayer (Cartesian Technologies) equipped with Chipmaker 3 quill pins (Telechem). Spotting volumes were about 0.5 nl resulting in a spot diameter of 120 µm with a pitch of 160 µm. Each transcript was spotted in duplicate 7.12 mm apart resulting in a total spotted area of 13.5x12.24 mm. After spotting, the slides were rehydrated by holding them over a bath of hot water (
70 °C), snap-dried on a 95–100 °C hot plate (5–10 s) and the DNA cross-linked using a UV cross-linker (150 mJ). The slides were soaked twice in 0.2% SDS for 2 min, twice in water for 2 min, and then transferred to boiling water for 2 min to allow DNA denaturation. After thorough drying (5 min), the slides were rinsed three times in 0.2% SDS for 1 min, once in water for 1 min, submerged in boiling water (2 s), and dried.
Sample preparation and labelling
mRNA was purified from RNA samples using oligo(dT) columns (AP Biotech). 2.5 µg of poly(A+) RNA was spiked with 1.0 ng of in vitro synthesized luciferase mRNA (Promega) and reverse transcribed in the presence of 5-(3-aminoallyl)-2'-dUTP (Sigma A0410) using 2 µg oligo(dT)21 as a primer. A 25 µl reaction containing, in addition to the oligo(dT)-annealed RNA template, 1 µl first strand buffer (Life Technologies), 10 mM DTT, 15 U ribonuclease inhibitor (Life Technologies), 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.3 mM dTTP, 0.2 mM aminoallyl-dUTP, and 150 U Superscript II RNase H-reverse transcriptase (Life Technologies) was incubated at 37 °C for 2 h. Nucleic acids were then ethanol-precipitated at room temperature and dissolved in 10 µl TE (10 mM TRIS–HCl, 1 mM EDTA, pH 8.0). Next, cDNA/mRNA hybrids were denatured (3 min at 98 °C) and chilled on ice. RNA was degraded by adding 2.5 µl 1 M NaOH and incubating 10 min at 37 °C. After adding 2.5 µl 1 M HEPES (pH 6.8) and 2.0 µl 1 M HCl, the cDNA was recovered by ethanol precipitation and resuspended in 10 µl 0.1 M sodium carbonate buffer (pH 9.3).
The modified cDNA was coupled to a fluorescent dye, either Cyanine 3 (Cy3) or Cyanine 5 (Cy5), using reactive Cy3- or Cy5-NHS-esters (AP Biotech). To this end 10 µl of 10 mM dye (in DMSO) was added to 10 µl of the cDNA sample and incubated at room temperature for 30 min. Finally, the labelled cDNA was ethanol precipitated twice and dissolved in 5 µl water.
Microarray hybridization
Following prehybridization at 42 °C for 2 h in a few ml of hybridization buffer (50% formamide, 5x Denhardt's reagent, 5x SSC, 0.2% SDS, 0.1 mg ml–1 denatured fish DNA), slides were rinsed in water and in isopropanol and then dried by centrifugation (1 min, 470 g). For a dual hybridization, 100 µl of hybridization mixture, containing both (Cy3- and Cy5-labelled) samples at a concentration corresponding to 8 ng (Cy3) or 2 ng (Cy5) of the initial mRNA per µl mixture, was used in order to obtain a comparable hybridization signal. A common reference sample was composed of all RNA samples under study. Prior to use, the hybridization mixture was heated at 95 °C (1 min), cooled on ice and spun down to remove any debris. Hybridizations were performed overnight at 42 °C using a Geneframe (15x16 mm, 65 µl volume; ABgene AB-0577) in a hybridization chamber. After hybridization, slides were washed at room temperature in 1x SSC, 0.1% SDS (5 min) followed by 0.1x SSC, 0.1% SDS (5 min) and rinsed briefly in 0.1x SSC before drying by centrifugation (1 min, 470 g).
To monitor gene expression patterns during senescence, a Cy5-labelled cDNA population was prepared from 0, 2, 4, and 7-d-old petal tissue (d0, d2, d4, and d7); from 2-d-old and 4-d-old ethylene-treated petal tissue (d2 ET and d4 ET); from 4, 7, and 10-d-old STS-treated tissue (d4 ST, d7 ST, and d10 ST); and from 4, 7, and 10-d-old sucrose-treated tissue (d4 SU, d7 SU, and d10 SU). For each hybridization, a single Cy3-labelled cDNA population prepared from pooled RNA samples was used as a reference.
Fragments from known carnation petal senescence-associated genes were included as controls: 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1=CARAO1; ten Have and Woltering, 1997), 1-aminocyclopropane-1-carboxylic acid synthase (ACS1=CARACC3; Park et al., 1992), ACS2 (=CARAS1; Henskens et al., 1994), SR5 (Woodson, 1994), SR8 (Meyer et al., 1991), SR12 (Raghothama et al., 1991), and S-adenosylmethionine (SAM) synthetase (Woodson et al., 1992).
A total of 2224 randomly selected (subtracted) PCR-amplified carnation cDNA fragments were spotted twice onto each glass slide. The population of fragments used had been enriched for flower-specific cDNAs by subtractive hybridization using stem tissue as the driver. After background subtraction, normalization, and evaluating the quality of the duplicates, the results from 1664 fragments (spotted in duplicate) remained available for further analysis. Only gene expression data resulting from the first experiment (first batch of flowers) are described here.
Data collection and normalization
Slides were scanned using a ScanArray 3000 (Packard BioScience) at 75% laser power and 75% attenuation at a resolution of 10 µm. The resulting Cy3 and Cy5 images were stored as TIFF-files. Total pixel intensities within a fixed area (circle diameter, 12 pixels) were obtained for each spot, using ArrayVision image analysis software (Imaging Research). For each hybridization experiment, average background values, calculated from the hybridization signals of the non-plant transcripts (negative controls), were subtracted to correct for non-specific fluorescence. Elements showing poor raw signals, that is not reaching 1.5x background, were filtered out. Normalization of the two samples in each hybridization was done using the mean hybridization signal of the full-length luciferase transcripts, resulting from the spiked luciferase mRNA. Finally, the expression ratios (R), that is, the expression in the sample under study compared with the reference sample, were calculated for each transcript. The reference sample consisted of a mixture of all RNA samples under study and was the same for every hybridization, allowing for direct comparison of all hybridization experiments. Expression ratios were calculated separately for the duplicates (R1, R2) and the average of both values was used for further analysis unless duplicates were poor (|2log(R1/R2)| >1), in which case the data were omitted. Next, the data of all experiments were combined and transcripts were omitted from further analysis when values were missing for four or more hybridizations out of the 12 of one set of experiments, or if values were missing from the time series of the same set (d0, d2, d4, and d7). This left a set of 1664 transcripts for further analysis. Hierarchical clustering was carried out on log-transformed data using GeneMaths software (Applied Maths, version 2.0), using Pearson correlation as a similarity measure.
Northern blotting and real-time PCR
13 µg of total RNA was used to make northern blots, according to standard procedure. Digoxigenin-labelled probes, consisting of the complete cloned cDNA fragment, were generated by PCR (PCR DIG Labeling Mix, Roche Diagnostics). Hybridizations were carried out under high-stringency conditions with ULTRAhyb solution (Ambion). Detection was done using CDP-Star (Ambion, Roche), following the manufacturer's instructions. For real-time PCR, cDNA-synthesis using Superscript reverse transcriptase (Gibco) was performed with 2 µg of total RNA and 1 µl Oligo (dT) (500 ng µl–1, Sigma). Amplification reactions, using the qPCR Core Kit for Sybr Green I (Eurogentec), contained 1 µl of (6x diluted) cDNA template and 10 pmol of each specific primer in a final volume of 40 µl. All qPCR reactions were performed in duplicate, using 0.5 µl 2000x diluted Sybr Green I solution (Molecular Probes). The amplification profile was 5 s at 95 °C, 20 s at 59 °C, and 40 s at 72 °C. PCR products were detected by monitoring the rise in fluorescence caused by Sybr Green I using the iCycler system (BIORAD).
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Time to visible senescence
Figure 1 shows the time to visible senescence (petal inrolling and wilting) of individual flowers. The average time to visible senescence of untreated flowers was 7.5 d. This period was decreased to 5.7 d after ethylene treatment (200 nl l–1; 18 h), and increased to 15.3 d by treatment with sucrose plus the antibacterial compound DICA (25 g l–1 and 150 mg l–1; continuously). Treatment with STS (0.2 mM; 18 h) resulted in desiccation of the flowers, without first showing inward movement of the petals and wilting. The time to desiccation was, on average, 26.6 d. Treatment with DICA alone only slightly increased the time to visible senescence.
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Microarray quality control
A total number of 2224 randomly selected PCR-amplified carnation cDNA fragments were spotted twice onto each microarray. After background subtraction, normalization, and evaluating the quality of the duplicates, the results from 1664 transcripts remained available for further analysis.
Seven cDNA fragments from known carnation petal senescence-associated genes had been included as controls. These controls all showed relative signal levels very similar to previously published data (Fig. 2A). Sequence analysis (see below) additionally revealed five ACO1/CARAO1 (encoding an ACC oxidase) and seven SR12-derived transcripts, all displaying the expected expression ratios.
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Microarray results for two selected transcripts were confirmed by northern blotting (Fig. 2B), and for three transcripts by real-time RT-PCR (Fig. 2C). An additional (biological) control was added by hybridizing a set of microarrays with labelled cDNA derived from a second independent experiment with a new batch of flowers. In these flowers, relative transcript levels during senescence were very similar to the results presented here. Only data resulting from the first experiment are further described here.
Expression of genes related to ethylene synthesis
Genes involved in ethylene synthesis can be used as markers for the expression of the transcripts here identified (Fig. 2). SAM synthetase is involved in the production of S-adenosylmethionine (SAM), the direct precursor of 1-aminocyclopropane-1-carboxylic acid (ACC). A transcript encoding SAM-synthetase increased sharply in abundance from day 0 to day 4, then decreased. One of the isolated ACC synthetase genes (ACS1) showed an exponential rise in transcript level, starting on day 0. It had apparently not yet reached its maximum on day 7. Ethylene treatment increased the transcript abundance of ACS1, whereas STS or sucrose treatment delayed or prevented this (Fig. 2A). This is similar to the effects of the treatments on the transcript abundance of the isolated ACC oxidase gene. This gene showed an exponential increase in transcript levels, from day 2 (Line figure in Fig. 2A).
Hierarchical clustering based on gene expression in untreated controls
Transcripts (in duplicate) of the non-treated controls were clustered on the basis of their expression patterns during the period of study (Fig. 3). The expression patterns after treatments with ethylene, STS, or sucrose are also shown in Fig. 3. The hierarchical clustering of these transcripts was the same as the one of the non-treated controls. Based on the sequence analysis results (see below), redundancy among the transcripts was estimated to be 35%. Transcripts that shared at least 50 bp overlap with more than 95% identity were defined as belonging to the same contig.
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In untreated flowers, only a relatively small number (approximately 4%) of the transcripts showed mRNA levels that became less abundant during senescence. A relatively small cluster (approximately 25% of all transcripts) displayed a transient and relatively small increase in mRNA abundance with a maximum at day 4. The largest cluster (around 65% of all spotted transcripts) showed a large increase in abundance throughout the 7 d of the experiment (Fig. 3). The expression pattern of most of the genes in this cluster resembled that of ACC oxidase, showing a large, exponential-like increase shortly prior to day 7.
Sequence analysis
268 transcripts were manually selected for sequence analysis, based on their expression pattern (both profile and amplitude). The summarized results from the BLASTX analysis are depicted in Table 1. Supplementary Table 1 contains additional information such as E-values, the percentage of amino acid identity, numerical expression data from our microarray analysis, and relevant references (see Supplementary Table 1 at JXB online).
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Several sequenced transcripts displayed homology to genes classified as (putative) transcription factors. Transcription factors included a NAC domain protein, a MADS-box factor, EIN3-like proteins, a MYB-like protein, a MYC protein, and Aux/IAA proteins. Putative signal transduction factors included a protein phosphatase 2C and a CBL-interacting protein kinase (CIPK) 6. The relative expression values of these transcripts are depicted in Fig. 4.
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Effects of ethylene, silver thiosulphate, and sugar
Compared with untreated flowers, the shifts in gene expression profiles observed after the various treatments were reminiscent of an earlier (sucrose and STS) or later (ethylene) developmental stage (Fig. 3).
Treatment with ethylene resulted in accelerated increases in transcript abundance of approximately 50% of all spotted transcripts belonging to the largest cluster. By contrast, the transcripts in the second largest cluster were not observed to show increased mRNA abundance following ethylene treatment.
Treatment with STS (an inhibitor of ethylene action) prevented or strongly delayed almost all of the changes in transcript abundance observed in the untreated control flowers (Fig. 3). Treatment with sucrose also prevented or delayed many changes in transcript abundance, very similar to STS (Fig. 3).
Figure 4A shows the expression data of some genes putatively involved in transcriptional regulation or in signal transduction. The expression patterns of two genes putatively involved in signal transduction are shown in Fig. 4B. These genes encoded a putative cytokinin oxidase and a putative CBL-interacting protein kinase (CIPK). The increase in expression levels were rather well correlated with the time to senescence, whereby ethylene advanced the increase in expression, and STS or sucrose delayed or prevented the increase in expression. Note the similarity with the expression profile of ACO1, the ACC oxidase gene used as a control (Fig. 2A).
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Discussion |
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Custom-made cDNA microarrays were used for transcript profiling during carnation petal senescence. The population of PCR fragments spotted on these microarrays was enriched for flower-specific cDNAs, using subtractive hybridization, a technique that allows for the isolation of differentially expressed cDNAs without a prior knowledge of their sequence. Approximately 94% of the fragments on the array displayed signal ratios that reflected changes in mRNA abundance, suggesting a strong effect of the subtraction procedure. Apparently, the use of (non-senescent) stem tissue as the ‘driver’ resulted in a microarray that was strongly enriched with petal-specific and senescence-specific transcripts
A few genes showed high expression at the first sampling point. Among these was a NAC-domain protein, which in control flowers became 8-fold down-regulated by day 2 of the experiment. NAC domain proteins belong to a large, plant-specific family of transcription factors. These have been implicated in a wide range of processes, including tolerance to biotic and abiotic stress, and programmed cell death in xylem tracheids and vessels (Kubo et al., 2005). An Arabidopsis NAC gene was expressed in leaves that were already senescent. Dark-induced leaf senescence in Arabidopsis was delayed when the gene was knocked out (Guo and Gan, 2006).
The transcript abundance of numerous genes increased during petal senescence. Up-regulation in one group of genes (the second largest cluster) was relatively low and occurred prior to day 4, after which down-regulation took place. In another group (the largest gene cluster) gene expression increased continuously, between day 0 and day 7, often with a large increase between days 4 and 7. After treatment with ethylene, about 50% of the genes in the largest cluster were up-regulated. Up-regulation in the largest cluster and the second largest cluster was prevented after STS treatment, showing that all genes in these two clusters were regulated by endogenous ethylene.
Early changes in transcription factors other than EIL
Transcripts encoding (putative) transcription factors that were up-regulated from day 0 included a putative MYB-like DNA binding protein, a zinc finger transcription factor, and a MYC-type DNA binding protein. The possible function of these genes in the regulation of senescence is as yet unclear. Another gene showing up-regulation from day 0 shared significant sequence similarity with genes encoding MADS-box proteins. Several MADS box transcriptions factors were down-regulated prior to cell death in barley seed endosperm (Sreenivasulu et al., 2006). In tomato, one such MADS-box factor (RIN) is critical to developmental control of fruit ripening (Vrebalov et al., 2002). The translated carnation fragment shared 34% identity with tomato RIN, but shared higher identity (55%) with the Arabidopsis PISTILLATA protein. Previously, a putative MADS-box gene related to Iris petal senescence was identified (van Doorn et al., 2003) that shared 51% identity with tomato RIN. The function of the MADS-box proteins in senescence is not known.
EIL genes
As described in the Introduction, EIN3-like (EIL) genes are pivotal in ethylene signalling. Three transcripts resembling EIL genes were identified. Two of these were part of Dc-EIL1 (Waki et al., 2001), whereas one represented Dc-EIL3 (Iordachescu and Verlinden, 2005). In the present tests, Dc-EIL1 was not much affected by any treatment. By contrast, a large increase in the expression of Dc-EIL3 was observed between day 4 and day 7 in senescent carnation petals. Most of the increase was hastened after ethylene treatment. This confirms the data of Iordachescu and Verlinden (2005). The increase was delayed after STS treatment, showing that endogenous ethylene is involved.
The gene expression data do not contradict the idea of a master switch during senescence, controlling the co-ordinated up-regulation of numerous ethylene-responsive genes. Dc-EIL3 might be (part of) this master switch.
Furthermore, the up-regulation of EIL was related to the up-regulation of the genes that result in ethylene synthesis (ACC synthase and ACC oxidase), both during senescence and after ethylene treatment (Fig. 2). As mentioned in the Introduction, ethylene treatment does not change the pattern of the autocatalytic rise in ethylene production, but advances it. The increase in EIL protein, induced by ethylene, might therefore explain, wholly or partially, the autocatalytic character of the senescence-associated ethylene production in carnation petals.
Hormones other than ethylene; proteasome degradation
Transcripts suggesting a role in senescence of hormones other than ethylene encoded Aux/IAA proteins, which are transcriptional repressors with a key function in regulating auxin-responsive genes (Bishopp et al., 2006), and a cytokinin oxidase/dehydrogenase, which degrades cytokinin (Taverner et al., 2000).
An F-box protein was up-regulated between day 0 and 2. The Arabidopsis genome contains about 700 F-box genes that function in targeting specific proteins for ubiquitin-dependent 26S proteasome-mediated protein degradation (Vierstra, 2003). One such F-box protein, ORE9, was shown to be required for the initiation of leaf senescence in Arabidopsis (Woo et al., 2001). Several other transcripts, all induced during carnation petal senescence, were homologous to genes encoding various other components of the 26S proteasome machinery, including RPT6, RPN2, a RING finger protein and a U-box containing protein. All were up-regulated between day 0 and day 2. The data support a role for 26S proteasome-mediated protein degradation during floral senescence.
Effect of sucrose
Sucrose treatment delayed the time to visible senescence. It is also known to delay the large rise in ethylene production. The effect of sucrose on gene expression, with the exception of a few genes, was identical to that of STS. The delay of visible senescence, after sucrose treatment, is therefore associated with preventing the up-regulation of numerous genes. Since these genes are shown to be regulated by ethylene, the data indicate that sucrose regulates ethylene signalling.
Sucrose treatment prevented the increase in transcript abundance of Dc-EIL3. It is tempting to speculate that endogenous levels of soluble sugars act as a regulator of flower senescence by influencing Dc-EIL3 gene expression. In addition, sucrose might promote EIL degradation in proteasomes, as observed in Arabidopsis (Yanagisawa et al., 2003). Sucrose loading of a cell might thus prevent the accumulation of one or more EIL proteins, because it promotes EIL degradation in proteasomes. The low level of EIL might then prevent the up-regulation of numerous genes, including the gene(s) encoding EIL.
Genes associated with remobilization
Breakdown of macromolecules into mobile compound is important in plant senescence (Buchanan-Wollaston et al., 2003). Numerous identified transcripts may be involved in the degradation of proteins, lipids, or carbohydrates, including complex cell wall carbohydrates.
One transcript was identical to a previously described ethylene-regulated cysteine protease in carnation petals that is believed to play a role in protein remobilization (Jones et al., 1995) and three more (up-regulated) transcripts most likely represented carnation cysteine protease genes. One of these cysteine protease transcripts displayed homology to a tobacco vacuolar processing enzyme (VPE), a caspase-like protein that has been associated with senescence and virus-induced hypersensitive cell death (Hatsugai et al., 2006).
A rather large number of transcripts might be associated with cell wall degradation. The most abundant transcript in our experiments (17 sequenced transcripts) was strongly homologous to an A. thaliana β-xylosidase gene. These transcripts were strongly up-regulated during carnation petal senescence. β-xylosidases have been implicated in senescence-related cell wall metabolism (Goujon et al., 2003). Eleven other putative β-xylosidase transcripts (three contigs) displayed a similar expression pattern. All these transcripts seem to be part of the carnation SR5 gene (EJ Woltering, unpublished results). SR5 has been previously described to encode a putative β-glucosidase (Woodson, 1994), but its sequence was never submitted to a public database. The present data thus indicate that SR5 rather encodes a putative β-xylosidase. Two transcripts are highly homologous to expansin encoding genes, involved in cell wall loosening during growth or disassembly (Cosgrove, 2000).
Genes associated with defence
Several transcripts, all up-regulated during senescence, might be associated with plant defence. These transcripts encoded enzymes putatively involved in isoprenoid biosynthesis, or encoded HSR201 (a putative alcohol acetyl transferase induced during the hypersensitive response to pathogen attack; Czernic et al., 1996), a thaumatin/PR5-like protein, a lipid-transfer-like protein, Avr9/Cf-9 rapidly elicited protein 146, salicylic acid-binding protein 2 (SABP2), and a polygalacturonase inhibitor-like leucine rich repeat (LRR) protein. It has previously been found that senescence of Iris petals was also accompanied by the induction of a considerable number of putative defence-related genes (van Doorn et al., 2003). Up-regulation of defence-related genes may, therefore, be characteristic for flower senescence, as is the case for leaf senescence (Bhalerao et al., 2003).
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Conclusions |
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Carnation petal senescence was associated with an increase in the expression of numerous genes. Several of the genes up-regulated at this time were putatively involved in the degradation of lipids, proteins, nucleic acids, and cell wall components. STS prevented the up-regulation of most of the senescence-associated genes, showing that their expression is regulated by endogenous ethylene. Sucrose treatment also strongly delayed the senescence-associated changes in gene expression. Sucrose treatment of cut carnation flowers resulted in the maintenance of otherwise decreasing levels of glucose, fructose, and sucrose in the petals (Nichols, 1975). It is therefore proposed that soluble sugars in the carnation petal cells act as a repressor of senescence at the transcriptional level.
EIL genes have been suggested to be important regulators in the ethylene signal transduction pathway. It was observed that the senescence-associated increase in Dc-EIL3 expression was delayed by STS and prevented by sucrose treatment. Sucrose, therefore, might negatively affect ethylene signalling, in part, by preventing the senescence-associated increase in EIL3 expression.
Apart from a possible role of EIL genes, it is not clear what regulates the changes in senescence-associated (and ethylene-inducible) gene expression. Several (putative) transcription factors were found that were down-regulated or up-regulated early during senescence. Their effect on ethylene signalling and gene expression is as yet unknown.
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For Supplementary Table 1 see JXB online. The table details the BLASTX analysis and relative expression values of the sequenced carnation cDNA transcripts. Transcripts were divided into 13 groups based on their (putative) function. Classification was based on the hierarchical clustering depicted in Fig. 4. The values given are 2log ratios of the expression levels relative to a common reference sample. Transcripts with E-values greater than 1x10–10 were considered to have no homology. Untreated samples: d0, d2, d4, d7; ethylene-treated sample: d2 ET, d4 ET; STS-treated samples: d4 ST, d7 ST, d10 ST; sucrose-treated samples: d4 SU, d7 SU, d10 SU; d=day.
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Acknowledgements |
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The authors thank Peter Balk and Adele van Houwelingen for technical assistance, and Ernst Woltering for critically reading the manuscript and helpful discussion. The work was supported by the Dutch Ministry of Agriculture, Nature Management and Food Quality. Work at Plant Research International was performed as part of the research programme of the Centre for BioSystems Genomics (CBSG) which is part of the Netherlands Genomics Initiative (NGI).
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* Present address: Department of Plant Systems Biology, VIB/Ghent University, Technologie Park 927, B-9052 Ghent, Belgium.
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Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research (1997) 25:3389–3402.
Bhalerao R, Keskitalo J, Sterky F, et al. Gene expression in autumn leaves. Plant Physiology (2003) 131:430–442.
Bishopp A, Mähönen AP, Helariutta Y. Signs of change: hormone receptors that regulate plant development. Development (2006) 133:1857–1869.
Buchanan-Wollaston VV, Earle S, Harrison E, Mathas E, Navabpour S, Page T, Pink D. The molecular analysis of leaf senescence: a genomics approach. Plant Biotechnology Journal (2003) 1:3–22.[CrossRef][Web of Science][Medline]
Chang S, Puryear J, Cairney J. A simple and efficient methode to isolate RNA from pine trees. Plant Molecular Biology Reporter (1993) 11:114–117.
Cosgrove DJ. New genes and new biological roles for expansins. Current Opinion in Plant Biology (2000) 3:73–78.[CrossRef][Web of Science][Medline]
Czernic P, Huang HC, Marco Y. Characterization of hsr201 and hsr515, two tobacco genes preferentially expressed during the hypersensitive reaction provoked by phytopathogenic bacteria. Plant Molecular Biology (1996) 31:255–265.[CrossRef][Web of Science][Medline]
Goujon T, Minic Z, El Amrani A, Lerouxel O, Aletti E, Lapierre C, Joseleau JP, Jouanin L. AtBXL1, a novel higher plant (Arabidopsis thaliana) putative beta-xylosidase gene, is involved in secondary cell wall metabolism and plant development. The Plant Journal (2003) 33:677–690.[CrossRef][Web of Science][Medline]
Guo H, Ecker JR. The ethylene signalling pathway: new insights. Current Opinion in Plant Biology (2004) 7:40–41.[CrossRef][Web of Science][Medline]
Guo Y, Gan S. ATNAP, a NAC family transcription factor, has an important role in leaf senescence. The Plant Journal (2006) 46:601–612.[CrossRef][Web of Science][Medline]
Hatsugai N, Kuroyanagi M, Nishimura M, Hara-Nishimura I. A cellular suicide strategy of plants: vacuole-mediated cell death. Apoptosis (2006) 11:905–911.[CrossRef][Web of Science][Medline]
Henskens JA, Rouwendal GJ, ten Have A, Woltering EJ. Molecular cloning of two different ACC synthase PCR transcripts in carnation flowers and organ-specific expression of the corresponding genes. Plant Molecular Biology (1994) 26:453–458.[CrossRef][Web of Science][Medline]
Iordachescu M, Verlinden S. Transcriptional regulation of three EIN3-like genes of carnation (Dianthus caryophyllus L. cv. Improved White Sim) during flower development and upon wounding, pollination, and ethylene exposure. Journal of Experimental Botany (2005) 56:2011–2018.
Jones M, Larsen PB, Woodson WR. Ethylene-regulated expression of a carnation cysteine proteinase during flower petal senescence. Plant Molecular Biology (1995) 28:505–512.[CrossRef][Web of Science][Medline]
Kubo M, Udagawa M, Nishikubo N, Horiguchi G, Yamaguchi M, Ito J, Mimura T, Fukuda H, Demura T. Transcription switches for protoxylem and metaxylem vessel formation. Genes and Development (2005) 19:1855–1860.
Mayak S, Dilley DR. Effect of sucrose on response of cut carnation to kinetin, ethylene and abscisic acid. Journal of the American Society for Horticultural Science (1976) 10:583–585.
Meyer RC, Goldsbrough PB, Woodson WR. An ethylene-responsive flower senescence-related gene from carnation encodes a protein homologous to glutathione S-transferases. Plant Molecular Biology (1991) 17:277–281.[CrossRef][Web of Science][Medline]
Nichols R. Senescence of the cut carnation flower: respiration and sugar status. Journal of Horticultural Science (1973) 48:111–121.
Nichols R. Senescence and sugar status of the cut flower. Acta Horticulturae (1975) 41:21–30.
Park KY, Drory A, Woodson WR. Molecular cloning of a 1-aminocyclopropane-1-carboxylate synthase from senescing carnation flower petals. Plant Molecular Biology (1992) 18:377–386.[CrossRef][Web of Science][Medline]
Price J, Laxmi A, St Martin SK, Jang JC. Global transcription profiling reveals multiple sugar signal transduction mechanisms in Arabidopsis. The Plant Cell (2004) 16:2128–2150.
Raghothama KG, Lawton KA, Goldsbrough PB, Woodson WR. Characterization of an ethylene-regulated flower senescence-related gene from carnation. Plant Molecular Biology (1991) 17:61–71.[CrossRef][Web of Science][Medline]
Sreenivasulu N, Radchuk V, Strickert M, Miersch O, Weschke W, Wobus U. Gene expression patterns reveal tissue-specific signalling networks controlling programmed cell death and ABA-regulated maturation in developing barley seeds. The Plant Journal (2006) 47:310–327.[CrossRef][Web of Science][Medline]
Taverner EA, Letham DS, Wang J, Cornish E. Inhibition of carnation petal inrolling by growth retardants and cytokinins. Australian Journal of Plant Physiology (2000) 27:357–362.[Web of Science]
ten Have A, Woltering EJ. Ethylene biosynthetic genes are differentially expressed during carnation (Dianthus caryophyllus L.) flower senescence. Plant Molecular Biology (1997) 34:89–97.[CrossRef][Web of Science][Medline]
van Doorn WG. Is petal senescence due to sugar starvation? Plant Physiology (2004) 134:35–42.
van Doorn WG, Balk PA, van Houwelingen AM, Hoeberichts FA, Hall RD, Vorst O, van der Schoot C, van Wordragen MF. Gene expression during anthesis and senescence in Iris flowers. Plant Molecular Biology (2003) 53:845–863.[CrossRef][Web of Science][Medline]
Vierstra RD. The ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins. Trends in Plant Science (2003) 8:135–142.[CrossRef][Web of Science][Medline]
Vrebalov J, Ruezinsky D, Padmanabhan V, White R, Medrano D, Drake R, Schuch W, Giovannoni J. A MADS-box gene necessary for fruit ripening at the tomato Ripening-inhibitor (Rin) locus. Science (2002) 296:343–346.
Waki K, Shibuya K, Yoshioka T, Hashiba T, Satoh S. Cloning of a cDNA encoding EIN3-like protein (DC-EIL1) and decrease in its mRNA level during senescence in carnation flower tissues. Journal of Experimental Botany (2001) 52:377–379.
Woo HR, Chung KM, Park JH, Oh SA, Ahn T, Hong SH, Jang SK, Nam HG. ORE9, an F-box protein that regulates leaf senescence in Arabidopsis. The Plant Cell (2001) 13:1779–1790.
Woodson WR. Molecular biology of flower senescence in carnation. In: Molecular and cellular aspects of plant reproduction—Scott RJ, Stead AD, eds. (1994) Cambridge: Cambridge University Press. 255–267.
Woodson WR, Lawton KA. Ethylene-induced gene expression in carnation petals. Plant Physiology (1988) 87:498–503.
Woodson WR, Park KY, Drory A, Larsen PB, Wang H. Expression of ethylene biosynthesis pathway transcripts in senescing carnation flowers. Plant Physiology (1992) 99:526–532.
Yanagisawa S, Yoo SD, Sheen J. Differential regulation of EIN3 stability by glucose and ethylene signalling in plants. Nature (2003) 425:521–525.[CrossRef][Medline]
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