- Research article
- Open Access
Upregulation of a tonoplast-localized cytochrome P450 during petal senescence in Petunia inflata
© Xu et al; licensee BioMed Central Ltd. 2006
- Received: 05 January 2006
- Accepted: 13 April 2006
- Published: 13 April 2006
Gene expression in Petunia inflata petals undergoes major changes following compatible pollination. Severe flower wilting occurs reproducibly within 36 hours, providing an excellent model for investigation of petal senescence and programmed cell death. Expression of a number of genes and various enzyme activities involved in the degradation and remobilization of macromolecules have been found to be upregulated during the early stages of petal senescence.
By performing differential display of cDNAs during Petunia inflata petal senescence, a highly upregulated gene encoding a cytochrome P450 was identified. Analysis of the complete cDNA sequence revealed that the predicted protein is a member of the CYP74C family (CYP74C9) and is highly similar to a tomato CYP74C allene oxide synthase (AOS) that is known to be active on 9-hydroperoxides. Cloning of the petunia genomic DNA revealed an intronless gene with a promoter region that carries signals found in stress-responsive genes and potential binding sites for Myb transcription factors. Transcripts were present at detectable levels in root and stem, but were 40 times more abundant in flowers 36 hours after pollination. Ethylene and jasmonate treatment resulted in transitory increases in expression in detached flowers. A protein fusion of the CYP74C coding region to a C-terminal GFP was found to be located in the tonoplast.
Though oxylipins, particularly jasmonates, are known to be involved in stress responses, the role of other products of CYP74 enzymes is less well understood. The identification of a CYP74C family member as a highly upregulated gene during petal senescence suggests that additional products of fatty acid metabolism may play important roles during programmed cell death. In contrast to the chloroplast localization of AOS proteins in the CYP74A subfamily, GFP fusion data indicates that the petunia CYP74C9 enzyme is in the tonoplast. This result suggests that the highly similar CYP74C enzymes that have been identified in two other Solanaceous plants may also be associated with the vacuole, an organelle known to have a prominent role in programmed cell death.
- Differential Display
- Compatible Pollination
- Petal Senescence
- Tonoplast Vesicle
Plant cell death occurs during the hypersensitive response [1, 2], response to environmental stress , senescence , and the development of plant tissues and organs [3, 5]. Among these phenomena, petal senescence is of interest both because of its importance to the horticultural industry as well as a model for programmed cell death (PCD). Petal senescence shares a hallmark feature of PCD, namely DNA fragmentation [6, 7]. In contrast, an early apoptotic event common in mammalian cells, the relocation of cytochrome c from the mitochondrial membrane space into the cytosol, was not detected as a signal for wilting of petunia petal tissues . Evidently some plant death processes do not necessarily require cytochrome c release as a signal. In Arabidopsis protoplasts, when death was induced by C2 ceramide, loss of mitochondrial membrane potential and cytochrome c release were observed early in the death process. However, when protoporphyrin IX was used as the induction signal, although a decrease in membrane potential occurred, cytochrome c release was not observed until after the Arabidopsis protoplasts had died . In most other studies of various types of plant PCD, cytochrome c release was observed during PCD, for example, in stressed cultured cells [9, 10], tapetal cells , proteasome-inhibited epidermal cells , and toxin-treated mesophyll cells .
Subtractive cloning and differential display have been used to identify a number of genes that are highly induced during senescence. Consistent with the profound effect of ethylene on floral senescence in ethylene-sensitive flowers, petal wilting is preceded by the up-regulation of both ACC synthase and ACC oxidase in both Petunia and carnation [14, 15]. Most other genes up-regulated during petal senescence that have been identified so far encode enzymes involved in the degradation and remobilization of macromolecules (reviewed in [5, 16]), including a thiol protease , acyl-CoA oxidase , glutathione-S-transferase , DNases and RNases [6, 20, 21], and lipoxygenases [22, 23]. Other upregulated genes that have been identified have unknown functions, such as a calmodulin-binding protein  and a zinc-finger DNA-binding protein .
In the self-incompatible species Petunia inflata, petal senescence can be triggered reproducibly by compatible pollination, with clear wilting symptoms appearing at 36 hours after compatible pollination (HACP) . This pollination-induced petal senescence not only minimizes environmental influences, but also provides an inducible system to clone and investigate up-regulated genes associated with petal cell death. Using the technique of differential display during pollination-induced petal senescence, an upregulated gene with similarity to the CYP74C subfamily of cytochrome P450s was identified. Sequence analysis indicates the predicted protein (designated CYP74C9 by the P450 nomenclature committee, identified here as PiCYP74C9) is most related to an unusual tomato allene oxide synthase that preferentially metabolizes 9-hydroperoxides . While some AOS proteins have been localized to the chloroplast envelope, analysis of transgenic plants carrying PiCyP74C9 fused to GFP indicate that the P. inflata protein is located in the tonoplast.
Isolation of Psrgenes
Morphologically, programmed cell death (PCD) can be divided into two distinct stages , condemned/latent stage and execution stage. In the condemned stage, no obvious morphological changes are visible and the duration is quite variable. At 24 hours after compatible self-pollination (HACP), the P. inflata flowers appear quite normal but are condemned; at 36 hours the flowers are quite wilted . To identify genes that are up-regulated during the condemned phase of petal senescence, differential display (DD) was used to compare mRNA expression profiles  from transcripts of young petals from flowers that just opened with those of senescing petals from flowers at 24 and 36 HACP. This comparison was to identify those genes which may be involved in early events (24 hours) during the condemned phase as well as gene involved in late events of senescence equivalent to the execution stage in apoptosis .
Using a 1:1 ratio of mRNA from petals at 24 HACP and 36 HACP, a cDNA library specific to the petal and enriched in senescence-related RNAs was constructed. The primary library was screened for the coding regions of Psr genes. Two independent clones contained a sequence similar to ACC oxidase, an enzyme involved in ethylene biosynthesis. Two other independent clones carried a sequence similar to a member of the plant CYP74 family, cytochrome P450s. These genes were termed petal senescence related (psr) genes, Psr1 (ACC oxidase) and Psr2 (encoding CYP74C9).
Construction of full-length cDNA of Psr2and characteristics of CYP74C9
Developmental and tissue-specific expression of Psr2
Among the different tissues tested, Psr2 was expressed at low levels constitutively in the root and stem and at much lower levels in other young tissues, including healthy leaves and tissues from unopened flowers (Fig. 7). A densitometry measurement shows that the induced level of Psr2 mRNA in the pollinated petals at 36 HACP is about 40-fold higher than the constitutive level in either root or stem (Fig. 7). Since treatments with ethylene and jasmonates have been shown to accelerate petal senescence in P. hybrida [35, 36], ethephon or MEJA was applied to detached flowers of P. inflata. Ethephon is a commonly used substitute for ethylene and easily converted into ethylene within plant cells . In the presence of 1 mM ethephon, P. inflata flowers wilted within 24 hours (data not shown), whereas with simple distilled water treatment, detached flowers could last more than 6 days (data not shown). MEJA treatment seemed to promote a change in petal color rather than physical wilting (data not shown). Both ethephon treatment and MEJA treatment (Fig. 7) caused a transient increase in Psr2 transcripts in detached flowers.
Characterization of Psr2 gene organization and promoter elements
Cellular localization of PiCYP74C9
PiCYP74C9 lacks any defined targeting signal; analysis by a variety of web-based localization informatics programs gave inconclusive and contradictory results. To investigate the location of PiCYP74C9, the full coding region was fused in-frame to an enhanced GFP  and a strong promoter  and introduced into Nicotiana tabacum by Agrobacterium-mediated transformation.
The identification of macromolecules that increase in abundance during petal senescence is critical for our understanding of this process. Although a number of genes have been cloned that are highly expressed in senescing petals, the signal transduction pathways remain poorly understood. In addition to PiCYP74C9, a second highly induced known gene that we also identified during our study encodes ACC oxidase, which is involved in ethylene biosynthesis. The identification of ACC oxidase is not surprising, given that ethylene is known to be important in signaling petunia floral senescence [52, 53]. The ethylene peak in P. inflata petal tissue starts from 18 HACP and peaks at 24 HACP . This ethylene surge lags behind the up-regulation of ACC oxidase mRNA (Psr1) which shows up-regulation starting after 12 HACP (Fig. 6). This timing supports the theory that de novo synthesis of ethylene is required for the increased ethylene level.
Our previous study  showed that the total amount of RNA decreased about 50% at 24 HACP. Up to 36 HACP, the steady-state levels of the Psr1 and Psr2 transcripts continue to increase despite collapse of the floral shape and decrease in total RNA and protein contents, suggesting that Psr1 and Psr2 transcripts are either continuously transcribed or protected from the large-scale degradation of RNA in the senescing petal. This indicates active regulation of petal senescence by the plant.
Both ethylene and MEJA induced expression of Psr2 in unpollinated petals. Jasmonates also evidently play a role in the regulation of the gene encoding LeAOS3, the CYP74C member most closely related to PiCYP74C9, as expression of LeAOS3 in roots did not occur in a tomato mutant insensitive to jasmonates . LeAOS3 transcripts were found in germinating seedlings and roots but not cotyledons, mature leaves, stems, nor flower buds; evidently senescing tissue was not tested . Our data suggests that it will be worthwhile to examine the expression of the genes encoding LeAOS3 and the similar StAOS3 protein in stressed tissues to find out whether all of these synthases are upregulated during programmed cell death. Likewise, it will be interesting to determine whether or not LeAOS3 and StAOS3 are targeted to the tonoplast.
Lipoxygenases, which act upon lipids to produce the hydroperoxide substrates utilized by CYP74 cytochrome P450s (Fig. 12), have been found in a variety of locations within the plant cell, including the vacuole [57, 58]. Some CYP74 proteins have an N-terminal chloroplast transit peptide and are associated with the chloroplast. In flax, tomato, and barley, the transit sequence has been shown to be functional as targeting signal for certain AOS proteins to chloroplasts [48–50]. A tomato AOS was targeted to the outer chloroplast envelope membrane, while an HPL was targeted to the inner membrane . However, other CYP74 members exist with no predictable location and await experimental determination. For example, only one of four predicted AOS genes in rice carries a putative chloroplast transit sequence . Not all CYP74A members are found in the chloroplast; the guayule CYP74C AOS (PaAOS) is associated with rubber particles .
Our results indicate that PiCYP74C9::GFP expressed in tobacco leaves is localized in tonoplasts but not in chloroplasts, mitochondria, nor peroxisomes. We presume that PiCYP74C9 is also located in the tonoplast in petunia during petal senescence. To our knowledge, this is the first example of a CYP74 subfamily member to be localized in the tonoplast. Both N-terminal  and C-terminal  GFP fusions with known tonoplast proteins have resulted in GFP labeling of the tonoplast. None of the computer programs we tested (Predotar, TargetP, Psort) predicted PiCYP74C9 to be located in either plastids, mitochondria, or the secretory pathway.
The tonoplast localization of PiCYP74C9 is intriguing given recent information about the important role of the vacuole in programmed cell death. While it has been known for some time that caspase activity is involved in both developmental and hypersensitive response cell death, only recently has the vacuolar processing enzyme been shown to exhibit caspase activity that is important for the execution phase of several types of PCD [62–64]. During senescence, cell contents can be recycled by digestion in vacuoles in the process of autophagy . In some types of developmental programmed cell death, including petal senescence, the final stages coincide with ultrastructural changes and permeabilization of the tonoplast and other membranes (reviewed in [5, 16, 66]). Confirmation of the allene oxide synthase activity of PiCYP74C9 on 9-hydroperoxides and identification of the products may give insights into the role of this protein in petal senescence. Future analyses of the PiCYP74C9 protein should also reveal the significance of its compartmentation in the tonoplast.
By the technique of differential display, we have identified a cytochrome P450 that is expressed at a level 40 times greater in senescing petals than in vegetative tissue. Upregulation occurs in response to compatible pollination, ethylene treatment, or jasmonate treatment. The petunia gene encodes a protein highly similar to a tomato CYP74C protein known to exhibit allene oxide synthase activity, preferentially on 9-hydroperoxides. Both a complete cDNA and genomic sequence of this single-copy gene have been obtained The promoter region of the petunia gene exhibits several motifs found in stress-responsive genes as well as binding sites for a petunia transcription factor. A C-terminal GFP fusion protein was located in the tonoplast, a compartment where CYP74 members have not previously been detected. Phylogenetic analysis indicates that the CYP74C subfamily may warrant future division into two groups, as more information becomes available about AOS and HPL enzymes acting on 9-hydroperoxides.
Plant materials, growth, and pollination
Two different P. inflata populations bearing different S alleles can be used to pollinate each other. A line termed P. inflata-1 was derived from seed originally received from Ken Sink (Michigan State). The other population, termed P-S-14, was provided by D. Maizonnier (Dijon, France), who obtained it from a South American source. Plants were grown at 23°C under 16 hr daylight and 8 hr darkness. The compatibility of P-S-14 pollen on P. inflata-1 was confirmed by seed production, while self-pollination of P. inflata-1 flowers did not result in seed set. Pollen from P-S-14 was used to pollinate P. inflata-1 on the day of flower opening. At pollination, the five stamens were removed from P. inflata-1 flowers to reveal the stigma and to avoid incompatible pollination.
Transgenic tobacco cv. Petit Havana plants containing the PiCYP74C9::GFP fusion that were used for cellular fractionation were grown in a growth room at 23°C under 16 hr light and 8 hr darkness for about one month before organelle isolation. For fluorescence microscopy observations, transgenic tobacco plants containing the PiCYP74C9::GFP fusion were grown in a greenhouse under natural illumination. Both GFP genes were under the control of the 35S promoter with AMV translation enhancer  in the PGTPV-Kan vector . Transgenic tobacco plants were produced as described by Kwok and Hanson .
Chemical treatments of detached flowers
Flowers were cut at the pedicel the day of opening. Twelve flowers were used in each treatment with pedicel placed in the chemical solution held in a 24-well tissue culture plate (Northeast Container Corporation, Dover, NH). The plate was left in the greenhouse with 16 hr light and 8 hr darkness. The stock solution for ethephon (Sigma Chemical, St. Louis, MO) was 1 mM in ddH2O. The stock solution for MEJA (Bedoukian Research Inc., Danbury, CT) was 100 mM in 95% ethanol. Stock solutions were kept at -80°C. The working solution was diluted from the stock solution in ddH2O before use.
RNA isolation and RNA blot analysis
Total RNA was extracted with TRIZOL reagent according to the manufacturer's instruction (GIBCO BRL). RNA amounts were determined by OD260. RNA electrophoresis (10 μg total RNA per lane) was carried out in formaldehyde denaturing gels as described (Sambrook et al. 1989), but the concentration of formaldehyde in the gel and running buffer was reduced to 0.7 M. RNA was transferred to Genescreen membrane (NEN Research Products, Boston, MA) in 20xSSC and hybridized at 65°C overnight in Church buffer (250 mM NaPO4, pH 7.2, 7% SDS, 1% BSA, 1 mM EDTA). Probe labeling was done with the random priming labeling kit DECAprimeII (Ambion, Austin, TX). After overnight hybridization, filters were washed twice with 0.2xSSC/0.1%SDS at 65°C.
Total RNA was extracted from 10 petals at 0, 24, and 36 HACP using TRIZOL reagent with the following modification from standard protocol. Before isopropanol precipitation, two rounds of ether extraction were added. After isopropanol precipitation, RNA was dissolved in ddH2O and precipitated in the presence of 2 mM LiCl at 4°C overnight. RNA concentration was determined by A260. For differential display, 1.5 μg total RNA from 0 HACP was compared with that of 24 or 36 HACP following the instructions for differential display provided by GenHunter (Brookline, MA). Reagents used in reverse transcription were from GIBCO BRL and the PCR buffer was from GenHunter. The primers for differential display were a generous gift from Dr. Mikhail Nasrallah.
DNA isolation and blot analysis
Genomic DNA was isolated according to a modified CTAB method . For DNA blot analysis, equal amounts of genomic DNA were digested with restriction enzymes, separated on a 1% agarose gel, and transferred to Hybond N+ (Amersham Pharmacia). The hybridization was done as described for RNA blot analysis.
cDNA library construction and screening
Total RNA isolation was performed following a phenol/SDS method  with modifications. Five grams of petals from 24 HACP or 36 HACP were ground in liquid N2 and further ground after adding 40 ml NES buffer (100 mM NaCl, 5 mM EDTA, 1% SDS) and 20 ml sodium acetate-buffered phenol (pH 4.0). The mixture was then homogenized with a Polytron (Brinkmann Instruments Inc., Westbury, NY) for 2 min. Messenger RNA was purified through an oligo(dT)-cellulose column (type 7) following the manufacturer's instruction (Amersham Pharmacia). Handling of the cDNA library, including construction, titering, screening, and in vivo excision, followed the manufacturer's instructions. The ZAP Express cDNA synthesis kit and ZAP Express cDNA Gigapack III gold cloning kit (Stratagene, La Jolla, CA) were used for the cDNA library construction. Starting with 5 μg mRNA from senescing petals (2.5 μg from 24 HACP and 2.5 μg from 36 HACP), a primary cDNA library with about 540,000 recombinant phage was obtained. About 25,000 primary phage were screened to clone the gene.
Tobacco leaves were homogenized in Hepes-NaOH (pH 7.5) containing 14 mM 2-mercaptoethanol and protease inhibitor cocktail (Complete Mini, Roche) in a chilled mortar and pestle. The homogenate was mixed with an equal volume of SDS sample buffer consisting of 200 mM Tris-HCl (pH 8.5), 2% (w/v) SDS, 0.7 M 2-mercaptoethanol, and 20% (v/v) glycerol and then it was boiled for 3 min or heated at 50°C for 20 min when used in comparison with tonoplast vesicles. Following microcentrifugation, the supernatant was taken for use as the whole leaf extract.
For the isolation of organelles, leaves were cut into small slices (approximately 1 mm× 10 mm) with a razor blade and homogenized in a Polytron for 2 sec five to seven times with 5 ml of a homogenizing buffer per gram fresh weight of leaves.
Chloroplasts were isolated from 30 g of leaves essential as described previously  except that the extraction and Percoll gradient buffer contained 2 mM EDTA instead of 10 mM EDTA. Intact chloroplasts were collected from a green layer near the bottom of Percoll gradients, suspended in 50 mM Hepes-NaOH (pH 8.0) containing 0.3 M mannitol and 2 mM EDTA, and centrifuged at 3,000 g for 40 s. The resulting pellet was used as the purified chloroplast fraction.
Mitochondria were isolated from 60 g of leaves using Percoll gradient centrifugation with 0–5% (w/v) PVP-40 preformed gradient based on the method described by Day et al with slight modifications as follows. Leaves were homogenized with a grinding buffer consisting of 25 mM Mops-KOH (pH 7.8) containing 0.4 M mannitol, 10 mM Tricine, 8 mM cysteine, 1 mM EGTA, 1% (w/v) PVP-40, and 0.1% (w/v) BSA. The homogenate was passed through eight layers cheesecloth and centrifuged at 1,000 g for 5 min. The supernatant was centrifuged again at 12,000 g for 15 min. The pellet was washed with 25 mM Mops-KOH (pH 7.2) containing 0.4 M mannitol and 1 mM EGTA and was suspended in the same buffer and layered on top of a solution of 28% (v/v) Percoll in 25 mM Mops-KOH (pH 7.2) containing 0.4 M mannitol and 0.1% (w/v) BSA with 0–5% (w/v) PVP-40 preformed gradient. After centrifugation at 40,000 g for 45 min, white band near the bottom of the tube was collected, suspended in 25 mM Mops-KOH (pH 7.2) containing 0.4 M mannitol and 1 mM EGTA, and centrifuged at 12,000 g for 15 min. The resulting pellet was used as the purified mitochondrial fraction.
Peroxisomes were isolated from 30 g of leaves based on the method described by Fukao et al.  as follows. Leaves were homogenized with a grinding buffer consisting of 20 mM pyrophosphate-HCl (pH 7.5) containing 0.3 M mannitol and 1 mM EDTA. The homogenate was passed through four layers of cheesecloth. The residue was homogenized with another grinding buffer again in a similar manner. The filtrates were combined and were centrifuged at 1,500 g for 10 min. The supernatant was centrifuged again at 10,000 g for 20 min. The pellet was washed with a grinding buffer, and was suspended in 4 ml of 10 mM Hepes-KOH (pH 7.2) containing 0.3 M mannitol and 1 mM EDTA and was subjected to centrifugation in Percoll. The suspension was layered on top of 5 ml of a 60% (v/v) and 30 ml of a 28% (v/v) solution of Percoll in 10 mM Hepes-KOH (pH 7.2) containing 1 mM EDTA and 0.3 M raffinose and was centrifuged at 40,000 g for 30 min without deceleration. After centrifugation, 1 ml fractions were collected by a fraction collector. The 6th fraction from the bottom, which had the highest catalase content judging from immunoblot analysis, was used as the purified peroxisome fraction.
Vacuoles were isolated from leaf protoplasts as follows. Leaves (15 g) were cut into small slices as described above and digested with an incubation medium conainting 1.5% Cellulase Onozuka RS, 0.4% Macerozyme R-10, 10 mM Mes-NaOH (pH 5.6), 8 mM CaCl2 and 0.7 M mannitol for 3 h at 30°C in darkness. After digestion, released protoplasts were passed through a 150-μm nylon mesh and washed with 0.7 M mannitol for three times. Protoplasts were treated with DEAE-dextran solution for the lysis and released vacuoles were separated by the discontinuous Ficoll gradient centrifugation as previously described by Asami et al. .
Tonoplast vesicles were isolated from 20 g of leaves based on the method described by Maeshima and Yoshida  as follows. Leaves were homogenized with a grinding buffer consisting of 50 mM Tris-acetate (pH 7.5) containing 0.25 M sorbitol, 2 mM EGTA, 1% (v/v) PVP-40, 2 mM DTT and 0.5 mM PMSF. The homogenate was passed through four layers of cheesecloth. The filtrates were centrifuged at 3,600 g for 10 min. The supernatant was centrifuged again at 120,000 g for 30 min. The pellet was suspended in 15 ml of 20 mM Tris-acetate (pH 7.5) containing 0.5 M sucrose, 1 mM EGTA, 2 mM DTT and 2 mM MgCl2 and poured into a centrifugation tube. The suspension was overlayed with 15 ml of 20 mM Tris-acetate (pH 7.5) containing 0.25 M sorbitol, 1 mM EGTA, 2 mM DTT and 2 mM MgCl2. After centrifugation at 120,000 g for 30 min in a Beckman 70 Ti rotor, the interference portion was collected and diluted in a three-times volume of 20 mM Tris-acetate (pH 7.5) containing 0.25 M sorbitol, 1 mM EGTA, 2 mM DTT and 2 mM MgCl2. The suspension was then centrifuged at 120,000 g for 30 min and the resulting pellet was used as the tonoplast preparation.
Purified organelles, except tonoplast vesicles, were mixed with an equal volume of SDS sample buffer as above and then boiled for 3 min. Tonoplast vesicles were denatured at 50°C for 20 min. Protein concentration was determined by RC DC Protein Assay (Bio-Rad) based on Lowry method  according to the manufacturer's instruction. BSA was used as standard. Protein amounts loaded on 12.5% (w/v) SDS-PAGE gels were 5 μg for whole leaf extract, 3 μg for chloroplasts, 0.3 μg for mitochondria and peroxisomes, and 0.15 μg for tonoplasts. Immunoblot analysis was performed by the method of Towbin et al. .
Antibodies used were rabbit anti-GFP antibodies (1:5,000, Molecular Probes), affinity-purified anti-large subunit of Rubisco antibodies (1:20,000; ) from rabbit anti-rice whole Rubisco antibodies  and rabbit anti-rice coupling factor 1 of chloroplastic ATPase antibodies (1:5,000; ), both gifts of Dr. Amane Makino; a mouse anti-maize mitochondrial ATPase alpha subunit antibody (1:200; ); a mouse anti-tobacco catalase monoclonal antibody purchased from Princeton University Molecular Biology Department Monoclonal Antibody Facility (1:500; ); anti-peptide antibody for vacuolar pyrophosphatase corresponding to the sequences HKAAVIGDTIGDPLK (putative loop XII, 1:50,000), a gift of Dr. Philip Rea ; and anti-binding protein, a gift of Dr. Eliot Herman (1:50,000, ).
Laser-scanning confocal and differential interference contrast microscopy was performed with a Leica TCS-SP2 confocal scanning head mounted on a Leica DMRE-7 (SDK) upright microscope equipped with a 100×HCX PL APO oil immersion objective (NA = 1.40; Leica Microsysytems Inc., Bannockburn, IL, USA). GFP was excited with the 488 nm line of a 4-line Argon ion laser and emission of GFP was detected between 500 and 581 nm.
We thank Dr. Mikhail Nasrallah for providing differential display primers and Dr. Ikeda Seishi for instruction in differential display. We are grateful to Rainer H. Köhler for valuable discussions and technical help, and thank Dr. Amane Makino (Tohoku University), Dr. Philip Rea (University of Pennsylvania), and Dr. Eliot Herman (USDA-ARS, Danforth Center) for gifts of antibodies. We thank Alex Wong for providing output from the PAUP program and Dr. John C. Robbins for assistance with sequence comparisons. This work was supported by a NSF/DOE/USDA training grant in Molecular Mechanisms of Plant Processes, DOE Energy Biosciences (DE FG02-89ER14030), and USDA CUAES funds.
- Nimchuk Z, Eulgem T, Holt BE, Dangl JL: Recognition and response in the plant immune system. Annual Review of Genetics. 2003, 37: 579-609.PubMedView ArticleGoogle Scholar
- Greenberg JT, Yao N: The role and regulation of programmed cell death in plant-pathogen interactions. Cell Microbiol. 2004, 6: 201-211.PubMedView ArticleGoogle Scholar
- Beers EP, McDowell JM: Regulation and execution of programmed cell death in response to pathogens, stress and developmental cues. Curr Opin Plant Biol. 2001, 4: 561-567.PubMedView ArticleGoogle Scholar
- Guo Y, Gan S: Leaf senescence: signals, execution, and regulation. Curr Top Dev Biol. 2005, 71: 83-112.PubMedView ArticleGoogle Scholar
- Rogers HJ: Cell death and organ development in plants. Curr Top Dev Biol. 2005, 71: 225-261.PubMedView ArticleGoogle Scholar
- Xu Y, Hanson MR: Programmed cell death during pollination-induced petal senescence in petunia. Plant Physiol. 2000, 122: 1323-1333.PubMedPubMed CentralView ArticleGoogle Scholar
- Orzaez D, Granell A: DNA fragmentation is regulated by ethylene during carpel senescence in Pisum sativum. Plant Journal. 1997, 11: 137-144.View ArticleGoogle Scholar
- Yao N, Eisfelder BJ, Marvin J, Greenberg JT: The mitochondrion--an organelle commonly involved in programmed cell death in Arabidopsis thaliana. Plant J. 2004, 40: 596-610.PubMedView ArticleGoogle Scholar
- Stein JC, Hansen G: Mannose induces an endonuclease responsible for DNA laddering in plant cells. Plant Physiol. 1999, 121: 71-79.PubMedPubMed CentralView ArticleGoogle Scholar
- Tiwari BS, Belenghi B, Levine A: Oxidative stress increased respiration and generation of reactive oxygen species, resulting in ATP depletion, opening of mitochondrial permeability transition, and programmed cell death. Plant Physiol. 2002, 128: 1271-1281.PubMedPubMed CentralView ArticleGoogle Scholar
- Balk J, Leaver CJ: The PET1-CMS mitochondrial mutation in sunflower is associated with premature programmed cell death and cytochrome c release. Plant Cell. 2001, 13: 1803-1818.PubMedPubMed CentralView ArticleGoogle Scholar
- Kim M, Ahn JW, Jin UH, Choi D, Paek KH, Pai HS: Activation of the programmed cell death pathway by inhibition of proteasome function in plants. J Biol Chem. 2003, 278: 19406-19415.PubMedView ArticleGoogle Scholar
- Curtis MJ, Wolpert TJ: The oat mitochondrial permeability transition and its implication in victorin binding and induced cell death. Plant J. 2002, 29: 295-312.PubMedView ArticleGoogle Scholar
- Tang XY, Woodson WR: Temporal and spatial expression of 1-aminocyclopropane-1-carboxylate oxidase mRNA following pollination of immature and mature petunia flowers. Plant Physiol. 1996, 112: 503-511.PubMedPubMed CentralGoogle Scholar
- Woodson WR, Park KY, Drory A, Larsen PB, Wang H: Expression of ethylene biosynthetic-pathway transcripts in senescing carnation flowers. Plant Physiol. 1992, 99: 526-532.PubMedPubMed CentralView ArticleGoogle Scholar
- Rubinstein B: Regulation of cell death in flower petals. Plant Mol Biol. 2000, 44: 303-318.PubMedView ArticleGoogle Scholar
- Jones ML, Larsen PB, Woodson WR: Ethylene-regulated expression of a carnation cysteine proteinase during flower petal senescence. Plant Mol Biol. 1995, 28: 505-512.PubMedView ArticleGoogle Scholar
- Do YY, Huang PL: Characterization of a pollination-related cDNA from Phalaenopsis encoding a protein which is homologous to human peroxisomal acyl-CoA oxidase. Arch Biochem Biophys. 1997, 344: 295-300.PubMedView ArticleGoogle Scholar
- Meyer RCJ, Goldsbrough PB, Woodson WR: An ethylene-responsive flower senescence-related gene from carnation encodes a protein homologous to glutathione S-transferases. Plant Mol Biol. 1991, 17: 277-281.PubMedView ArticleGoogle Scholar
- Taylor CB, Bariola PA, delCardayre SB, Raines RT, Green PJ: RNS2: a senescence-associated RNase of Arabidopsis that diverged from the S-RNases before speciation. Proc Natl Acad Sci U S A. 1993, 90: 5118-5122.PubMedPubMed CentralView ArticleGoogle Scholar
- Panavas T, Walker EL, Rubinstein B: Possible involvement of abscisic acid in senescence of daylily petals. J Exp Bot. 1998, 49: 1987-1997.View ArticleGoogle Scholar
- Peary JS, Prince TA: Floral lipoxygenase - activity during senescence and inhibition by phenidone. J Am Soc Hortic Sci. 1990, 115: 455-457.Google Scholar
- Panavas T, Rubinstein B: Oxidative events during programmed cell death of daylily (Hemerocallis hybrid) petals. Plant Science. 1998, 133: 125-138.View ArticleGoogle Scholar
- Yang T, Poovaiah BW: An early ethylene up-regulated gene encoding a calmodulin-binding protein involved in plant senescence and death. J Biol Chem. 2000, 275: 38467-38473.PubMedView ArticleGoogle Scholar
- van Der Krol AR, van Poecke RM, Vorst OF, Voogt C, van Leeuwen W, Borst-Vrensen TW, Takatsuji H, van Der Plas LH: Developmental and wound-, cold-, desiccation-, ultraviolet-B-stress-induced modulations in the expression of the petunia zinc finger transcription factor gene ZPT2-2. Plant Physiol. 1999, 121: 1153-1162.PubMedPubMed CentralView ArticleGoogle Scholar
- Itoh A, Schilmiller AL, McCaig BC, Howe GA: Identification of a jasmonate-regulated allene oxide synthase that metabolizes 9-hydroperoxides of linoleic and linolenic acids. J Biol Chem. 2002, 277: 46051-46058.PubMedView ArticleGoogle Scholar
- Earnshaw WC: Apoptosis: lessons from in vitro systems. Trends Cell Biol. 1995, 5: 217-220.PubMedView ArticleGoogle Scholar
- Liang P, Pardee AB: Differential display - A general protocol. Molecular Biotechnology. 1998, 10: 261-267.PubMedView ArticleGoogle Scholar
- Frohman MA, Dush MK, Martin GR: Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988, 85: 8998-9002.PubMedPubMed CentralView ArticleGoogle Scholar
- Song WC, Brash AR: Purification of an allene oxide synthase and identification of the enzyme as a cytochrome-P-450. Science. 1991, 253: 781-784.PubMedView ArticleGoogle Scholar
- Bate NJ, Sivasankar S, Moxon C, Riley JMC, Thompson JE, Rothstein SJ: Molecular characterization of an Arabidopsis gene encoding hydroperoxide lyase, a cytochrome P-450 that is wound inducible. Plant Physiol. 1998, 117: 1393-1400.PubMedPubMed CentralView ArticleGoogle Scholar
- Itoh A, Howe GA: Molecular cloning of a divinyl ether synthase. Identification as a CYP74 cytochrome P-450. J Biol Chem. 2001, 276: 3620-3627.PubMedView ArticleGoogle Scholar
- Szczesna-Skorupa E, Straub P, Kemper B: Deletion of a conserved tetrapeptide, PPGP, in P450 2C2 results in loss of enzymatic activity without a change in its cellular location. Arch Biochem Biophys. 1993, 304: 170-175.PubMedView ArticleGoogle Scholar
- Werck-Reichhart D, Bak S, Paquette S: Cytochromes P450. The Arabidopsis Book. 2002, 1-28.Google Scholar
- Porat R, Halevy AH, Serek M, Borochov A: An increase in ethylene sensitivity following Pollination is the initial event triggering an increase in ethylene production and enhanced senescence of Phalaenopsis orchid flowers. Physiologia Plantarum. 1995, 93: 778-784.View ArticleGoogle Scholar
- Porat R, Reiss N, Atzorn R, Halevy AH, Borochov A: Examination of the possible involvement of lipoxygenase and jasmonates in pollination-induced senescence of Phalaenopsis and Dendrobium orchid flowers. Physiologia Plantarum. 1995, 94: 205-210.View ArticleGoogle Scholar
- Woltering EJ, Devrije T: Ethylene - a tiny molecule with great potential. Bioessays. 1995, 17: 287-290.View ArticleGoogle Scholar
- Coleman CE, Kao TH: The flanking R-regions of 2 Petunia inflata S-alleles are heterogeneous and contain repetitive sequences. Plant Mol Biol. 1992, 18: 725-737.PubMedView ArticleGoogle Scholar
- Shinshi H, Usami S, Ohmetakagi M: Identification of an ethylene-responsive region in the promoter of a tobacco class-I chitinase gene. Plant Mol Biol. 1995, 27: 923-932.PubMedView ArticleGoogle Scholar
- Maxson JM, Woodson WR: Cloning of a DNA-binding protein that interacts with the ethylene-responsive enhancer element of the carnation GST1 gene. Plant Mol Biol. 1996, 31: 751-759.PubMedView ArticleGoogle Scholar
- Oh SA, Lee SY, Chung IK, Lee CH, Nam HG: A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence. Plant Mol Biol. 1996, 30: 739-754.PubMedView ArticleGoogle Scholar
- Martin C, PazAres J: MYB transcription factors in plants. Trends in Genetics. 1997, 13: 67-73.PubMedView ArticleGoogle Scholar
- Solano R, Nieto C, Avila J, Canas L, Diaz I, Paz-Ares J: Dual DNA binding specificity of a petal epidermis-specific MYB transcription factor (MYB.Ph3) from Petunia hybrida. EMBO J. 1995, 14: 1773-1784.PubMedPubMed CentralGoogle Scholar
- Joshi CP: An inspection of the domain between putative TATA Box and translation start site in 79 plant genes. Nucleic Acids Research. 1987, 15: 6643-6653.PubMedPubMed CentralView ArticleGoogle Scholar
- Kohler RH, Cao J, Zipfel WR, Webb WW, Hanson MR: Exchange of protein molecules through connections between higher plant plastids. Science. 1997, 276: 2039-2042.PubMedView ArticleGoogle Scholar
- Datla RSS, Bekkaoui F, Hammerlindl JK, Pilate G, Dunstan DI, Crosby WL: Improved high-level constitutive foreign gene-expression in plants using an AMV RNA4 untranslated leader sequence. Plant Science. 1993, 94: 139-149.View ArticleGoogle Scholar
- Reisen D, Leborgne-Castel N, Ozalp C, Chaumont F, Marty F: Expression of a cauliflower tonoplast aquaporin tagged with GFP in tobacco suspension cells correlates with an increase in cell size. Plant Mol Biol. 2003, 52: 387-400.PubMedView ArticleGoogle Scholar
- Froehlich JE, Itoh A, Howe GA: Tomato allene oxide synthase and fatty acid hydroperoxide lyase, two cytochrome P450s involved in oxylipin metabolism, are targeted to different membranes of chloroplast envelope. Plant Physiol. 2001, 125: 306-317.PubMedPubMed CentralView ArticleGoogle Scholar
- Harms K, Atzorn R, Brash A, Kuhn H, Wasternack C, Willmitzer L, Penacortes H: Expression of a flax allene oxide synthase cDNA leads to increased endogenous jasmonic acid (JA) levels in transgenic potato plants but not to a corresponding activation of JA-responding genes. Plant Cell. 1995, 7: 1645-1654.PubMedPubMed CentralView ArticleGoogle Scholar
- Maucher H, Hause B, Feussner I, Ziegler J, Wasternack C: Allene oxide synthases of barley (Hordeum vulgare cv. Salome): tissue specific regulation in seedling development. Plant Journal. 2000, 21: 199-213.PubMedView ArticleGoogle Scholar
- Rea PA, Britten CJ, Sarafian V: Common identity of substrate-binding subunit of vacuolar H+-translocating inorganic pyrophosphatase of higher plant cells. Plant Physiol. 1992, 100: 723-732.PubMedPubMed CentralView ArticleGoogle Scholar
- Jones ML, Chaffin GS, Eason JR, Clark DG: Ethylene-sensitivity regulates proteolytic activity and cysteine protease gene expression in petunia corollas. J Exp Bot. 2005, 56: 2733-2744.PubMedView ArticleGoogle Scholar
- Langston BJ, Bai S, Jones ML: Increases in DNA fragmentation and induction of a senescence-specific nuclease are delayed during corolla senescence in ethylene-insensitive (etr1-1) transgenic petunias. J Exp Bot. 2005, 56: 15-23.PubMedView ArticleGoogle Scholar
- Singh A, Evensen KB, Kao TH: Ethylene synthesis and floral senescence following compatible and incompatible pollinations in Petunia inflata. Plant Physiol. 1992, 99: 38-45.PubMedPubMed CentralView ArticleGoogle Scholar
- Schuler MA, Werck-Reichhart D: Functional genomics of P450s. Annual Review of Plant Biology. 2003, 54: 629-667.PubMedView ArticleGoogle Scholar
- Nelson DR: Cytochrome P450 and the individuality of species. Archives of Biochemistry and Biophysics. 1999, 369: 1-10.PubMedView ArticleGoogle Scholar
- Fischer AM, Dubbs WE, Baker RA, Fuller MA, Stephenson LC, Grimes HD: Protein dynamics, activity and cellular localization of soybean lipoxygenases indicate distinct functional roles for individual isoforms. Plant J. 1999, 19: 543-554.PubMedView ArticleGoogle Scholar
- Tranbarger TJ, Franceschi VR, Hildebrand DF, Grimes HD: The soybean 94-kilodalton vegetative storage protein is a lipoxygenase that is localized in paraveinal mesophyll cell vacuoles. Plant Cell. 1991, 3: 973-987.PubMedPubMed CentralView ArticleGoogle Scholar
- Haga K, Iino M: Phytochrome-mediated transcriptional up-regulation of allene oxide synthase in rice seedlings. Plant Cell Physiol. 2004, 45: 119-128.PubMedView ArticleGoogle Scholar
- Pan Z, Durst F, Werck-Reichhart D, Gardner HW, Camara B, Cornish K, Backhaus RA: The major protein of guayule rubber particles is a cytochrome P450. Characterization based on cDNA cloning and spectroscopic analysis of the solubilized enzyme and its reaction products. J Biol Chem. 1995, 270: 8487-8494.PubMedView ArticleGoogle Scholar
- Uemura T, Yoshimura SH, Takeyasu K, Sato MH: Vacuolar membrane dynamics revealed by GFP-AtVam3 fusion protein. Genes Cells. 2002, 7: 743-753.PubMedView ArticleGoogle Scholar
- Kuroyanagi M, Yamada K, Hatsugai N, Kondo M, Nishimura M, Hara-Nishimura I: Vacuolar processing enzyme is essential for mycotoxin-induced cell death in Arabidopsis thaliana. J Biol Chem. 2005, 280: 32914-32920.PubMedView ArticleGoogle Scholar
- Hara-Nishimura I, Hatsugai N, Nakaune S, Kuroyanagi M, Nishimura M: Vacuolar processing enzyme: an executor of plant cell death. Curr Opin Plant Biol. 2005, 8: 404-408.PubMedView ArticleGoogle Scholar
- Lam E: Vacuolar proteases livening up programmed cell death. Trends Cell Biol. 2005, 15: 124-127.PubMedView ArticleGoogle Scholar
- Thompson AR, Vierstra RD: Autophagic recycling: lessons from yeast help define the process in plants. Curr Opin Plant Biol. 2005, 8: 165-173.PubMedView ArticleGoogle Scholar
- van Doorn WG, Woltering EJ: Many ways to exit? Cell death categories in plants. Trends Plant Sci. 2005, 10: 117-122.PubMedView ArticleGoogle Scholar
- Becker D, Kemper E, Schell J, Masterson R: New plant binary vectors with selectable markers located proximal to the left T-DNA border. Plant Mol Biol. 1992, 20: 1195-1197.PubMedView ArticleGoogle Scholar
- Kwok EY, Hanson MR: GFP-labelled Rubisco and aspartate aminotransferase are present in plastid stromules and traffic between plastids. J Exp Bot. 2004, 55: 595-604.PubMedView ArticleGoogle Scholar
- Fulton TM, Chunwongse J, Tanskley SD: Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol Biol Rep. 1995, 13: 207-209.View ArticleGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: A laboratory manual, 2nd Edn. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press; 1989.Google Scholar
- Hegeman CE, Hayes ML, Hanson MR: Substrate and cofactor requirements for RNA editing of chloroplast transcripts in Arabidopsis in vitro. Plant J. 2005, 42: 124-132.PubMedView ArticleGoogle Scholar
- Day DA, Neuburger M, Douce R: Biochemical chalacterization of chlorophyll-free mitochondria from pea leaves. Aust J Plant Physiol. 1985, 12: 219-228.View ArticleGoogle Scholar
- Fukao Y, Hayashi M, Nishimura M: Proteomic analysis of leaf peroxisomal proteins in greening cotyledons of Arabidopsis thaliana. Plant and Cell Physiology. 2002, 43: 689-696.PubMedView ArticleGoogle Scholar
- Asami S, Haranishimura I, Nishimura M, Akazawa T: Translocation of photosynthates into vacuoles in spinach leaf protoplasts. Plant Physiol. 1985, 77: 963-968.PubMedPubMed CentralView ArticleGoogle Scholar
- Maeshima M, Yoshida S: Purification and properties of vacuolar membrane proton-translocating inorganic pyrophosphatase from mung bean. J Biol Chem. 1989, 264: 20068-20073.PubMedGoogle Scholar
- Lowry OHRNJFALRRJ: Protein measurement with the folin phenol reagent. J Biol Chem. 1951, 193: 265-275.PubMedGoogle Scholar
- Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979, 76: 4350-4354.PubMedPubMed CentralView ArticleGoogle Scholar
- Ishida H, Nishimori Y, Sugisawa M, Makino A, Mae T: The large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase is fragmented into 37-kDa and 16-kDa polypeptides by active oxygen in the lysates of chloroplasts from primary leaves of wheat. Plant Cell Physiol. 1997, 38: 471-479.PubMedView ArticleGoogle Scholar
- Makino A, Mae T, Ohira K: Photosynthesis and ribulose 1,5-bisphosphate carboxylase in rice leaves - changes in photosynthesis and enzymes involved in carbon assimilation from leaf development through senescence. Plant Physiol. 1983, 73: 1002-1007.PubMedPubMed CentralView ArticleGoogle Scholar
- Hidema J, Makino A, Mae T, Ojima K: Photosynthetic characteristics of rice leaves aged under different irradiances from full expansion through senescence. Plant Physiol. 1991, 97: 1287-1293.PubMedPubMed CentralView ArticleGoogle Scholar
- Conley CA, Hanson MR: Tissue-specific protein expression in plant mitochondria. Plant Cell. 1994, 6: 85-91.PubMedPubMed CentralView ArticleGoogle Scholar
- Collings DA, Harper JDI, Marc J, Overall RL, Mullen RT: Life in the fast lane: actin-based motility of plant peroxisomes. Can J Bot. 2002, 80: 430-441.View ArticleGoogle Scholar
- Kalinski A, Rowley DL, Loer DS, Foley C, Buta G, Herman EM: Binding-protein expression Is subject to temporal, developmental and stress-induced regulation in terminally differentiated soybean organs. Planta. 1995, 195: 611-621.PubMedView ArticleGoogle Scholar
- Swofford D: PAUP*. Phylogenetic analysis using parsimony(*and other methods). 4th edition. Sunderland, Mass., Sinauer Associates; 2002.Google Scholar
- Howe GA, Schilmiller AL: Oxylipin metabolism in response to stress. Curr Opin Plant Biol. 2002, 5: 230-236.PubMedView ArticleGoogle Scholar
- Feussner I, Wasternack C: The lipoxygenase pathway. Annu Rev Plant Biol. 2002, 53: 275-297.PubMedView ArticleGoogle Scholar
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