JXB Advance Access originally published online on November 28, 2003
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Journal of Experimental Botany, Vol. 55, No. 394, pp. 59-68, January 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
Molecular analysis of programmed cell death during senescence in Arabidopsis thaliana and Brassica oleracea: cloning broccoli LSD1, Bax inhibitor and serine palmitoyltransferase homologues
Received 6 February 2003; Accepted 3 October 2003
1 The New Zealand Institute for Crop and Food Research Limited, Food Industry Science Centre, Private Bag 11 600, Palmerston North, New Zealand
* Present address: Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK. To whom correspondence should be sent. Fax: +64 6 351 7050. E-mail: easonj{at}crop.cri.nz
DNA sequences: The nucleotide sequence data reported will appear in the GenBank Nucleotide Sequence Databases under accession numbers: AF453320 (BoBI-1), AF453321 (BoBI-2), AF453322 (BoLSD1), AF453323 (BoBLSD2), AF525281 (BoSPT1), AF525282 (BoSPT2), and AF513990 (18S rRNA).
Abbreviations: BI, Bax inhibitor; DALE, days after leaf emergence; DAPI, 4,6-diamidino-2-phenylindole; HR, hypersensitive response; LSD1, lesion simulating disease; PCD, programmed cell death; SPT, serine palmitoyltransferase; TUNEL, TdT-mediated dUTP nick end labelling.
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Abstract |
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This study was undertaken to characterize the programmed cell death (PCD) processes that occur during detached and natural on-plant senescence and correlate them with the expression of putative regulatory genes that may be involved in the process. DNA fragmentation and TUNEL analysis of broccoli florets showed that DNA was processed into fragments of approximately 180 bp after 48 h of harvest-induced tissue senescence. Characteristic laddering patterns were also visible in Arabidopsis leaves undergoing natural on-plant senescence and during detached senescence. Several recently isolated plant proteins have been assigned a PCD role, for example, the zinc finger containing protein, LSD1 (lesion simulating disease); Bax inhibitor (BI); and serine palmitoyltransferase (SPT), an enzyme in the sphingolipid signalling pathway. Two cDNAs encoding each of these proteins were isolated from broccoli (BoBI-1, BoBI-2, BoLSD1, BoLSD2, BoSPT1, BoSPT2), and the mRNAs increased during harvest-induced senescence in floret tissue. Expression of the Arabidopsis homologues (AtBI-1, AtLSD1, AtSPT1) were also characterized during detached leaf senescence in Arabidopsis leaves. AtBI-1 expression was constitutively expressed during detached senescence, AtLSD1 expression remained constitutively low, and AtSPT1 expression increased during detached senescence.
Key words: Ceramide, DNA laddering, post-harvest, sphingolipid biosynthesis, TUNEL analysis.
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Introduction |
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Senescence is the final stage of an organ’s development that marks the transition from net carbon and nitrogen assimilation to a period of catabolism followed by nutrient diversion to the developing sinks (Himelblau and Amasino, 2001). Senescence is a programmed event responding to external and internal signals, it requires de novo gene expression and protein synthesis, and is controlled in a highly co-ordinated manner (Buchanan-Wollaston, 1997; Chandlee, 2001). Many genes have been isolated that show enhanced expression during senescence, and the sequencing and characterization of these has made a valuable contribution to the understanding of the processes that occur during senescence (Gan and Amasino, 1997). However, despite such advances, much remains to be discovered about the regulatory signals and pathways that control this process.
Programmed cell death (PCD) is also a genetically defined process associated with common morphological and biochemical changes. It is well established that PCD is an intrinsic part of the life cycle of all multicellular organisms studied so far, including both animals and plants (Pennell and Lamb, 1997). This cell-suicide process has been extensively studied in animals and can be initiated by a variety of stimuli, including developmental signals and environmental cues (Vaux and Strasser, 1996; Vaux and Korsmeyer, 1999). PCD is also critical for normal development, maintenance of tissue homeostasis, and for defence responses in plants (reviewed in Plant Molecular Biology, Vol. 44).
The current view is that the process of senescence and the process of PCD during senescence are under the control of a co-ordinated signalling pathway, consistent with the view that senescence involves PCD (Gan and Amasino, 1997). The most widely studied PCD is animal cell apoptosis, which is characterized by a distinct set of morphological and biochemical features that include cell shrinkage, cytoplasmic membrane blebbing, nuclei lobing, DNA fragmentation into characteristic ‘ladders’, and disassembly into apoptotic bodies (Pennell and Lamb, 1997). At the molecular level, extensive studies have revealed that pro-apoptotic stimuli deactivate the suppression of PCD by anti-apoptotic molecules (Bossey-Wetzel and Green, 1999). A cascade of cysteine-proteases (the caspases) is activated in a step-wise manner and acts on downstream substrates causing morphological changes and cell death (Salvesen, 1999).
The plant response to attempted infection by microbial pathogens is often accompanied by rapid cell death in and around the initial infection site, a reaction known as the hypersensitive response (HR). This response restricts pathogen growth and represents a form of PCD (Pennell and Lamb, 1997). Recent molecular studies provide functional evidence for the conservation of regulatory mechanisms in a plant’s response to pathogens, and the activation of PCD in other plant and animal systems. In animals, the mitochondrion integrates diverse cellular stress signals and initiates the death execution pathway, and studies indicate a similar involvement for mitochondria in regulating PCD in plants (Jones, 2000). But many of the cell-death regulators that have been characterized in humans, worms and flies are absent from the Arabidopsis genome, indicating that plants probably use other regulators to control the PCD process (Lam et al., 2001).
Very few regulators of PCD have been cloned from plants and shown to play an unequivocal role in the process. One of the more intriguing discoveries is that certain plant disease resistance (R) genes encode a ‘nucleotide-binding site plus leucine-rich repeat’ (NB-LRR), these protein domains have some homology with eukaryotic cell death effectors such as Apaf-1 and Ced4 (Dangl and Jones, 2001). Other genes that may have a role in regulatory PCD in plants include cysteine proteases (Eason et al., 2002b; Gan and Amasino, 1995; Drake et al., 1996); the zinc finger protein, LSD1 (Dietrich et al., 1997); Bax inhibitor proteins (Kawai et al., 1999; Sanchez et al., 2000); and proteins involved in sphingolipid and ceramide production, such as serine palmitoyltransferase (Birch et al., 1999). LSD1 (lesion simulating disease) was characterized first as a mutant exhibiting lesions in the absence of disease organisms (Dietrich et al., 1994) and was subsequently found to encode a zinc finger protein with homology to GATA-type transcription factors. It has been suggested that the LSD1 protein functions either negatively to regulate a pro-death pathway component or to activate a repressor of plant cell death (Dietrich et al., 1997). The Bax inhibitor (BI) protein was first isolated from humans using a yeast screen for proteins that prevented Bax-induced lethality, and was termed Bax Inhibitor-1 (Xu and Reed, 1998). Homologues of this protein have now been isolated from Arabidopsis (Sanchez et al., 2000), rice (Kawai et al., 1999), and barley (Huckelhoven et al., 2001).
Sphingolipid biochemistry is connected to both plant and animal PCD, as the sphinganine-analogue mycotoxins, fumonisin B1 and AAL toxin, which competitively inhibit ceramide biosynthesis in vitro and induce PCD in both plant and animal systems (Gilchrist, 1997). Serine palmitoyltransferase (SPT) catalyses the first committed step in the synthesis of sphingolipids and has recently been shown to regulate de novo ceramide generation during apoptosis (Perry et al., 2000). One of the first plant SPT genes was isolated from potato tubers infected with Phytophthora infestans, and gene expression was induced in the early stages of the hypersensitive response (Birch et al., 1999).
Research on broccoli floret senescence has led to the isolation of five different cysteine protease genes, which are differentially expressed during harvest-induced floret senescence (Coupe et al., 2003; BoCP5 unpublished data). In addition, it has recently been shown that DNA is processed into ladders during harvest-induced senescence of asparagus spears (Eason et al., 2002a). The current study was undertaken to determine whether other putative regulators of PCD (i.e. LSD1, BI, SPT) are expressed during tissue senescence, focusing on harvest-induced (or detached) senescence. Broccoli florets and Arabidopsis leaves have been used as model systems to determine whether the expression of these putative regulators of PCD also correlates with PCD-associated DNA processing. These data will increase knowledge of the pathways that may be involved in regulating the plant senescence process.
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Materials and methods |
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Plant material
Broccoli (Brassica oleracea var. italica) was harvested in the early morning (08.00 h) from a commercial property, Palmerston North, New Zealand. The 0 h floret samples were shaved off harvested heads with a razor blade and frozen in liquid nitrogen whilst in the field. The other heads were placed on ice. Once back in the laboratory, the broccoli heads were dissected, and branchlets of uniform size and development and from a similar position in the head were selected. Triplicate branchlets were held dry in the dark at a constant temperature of 20 °C for 12, 24, 48, 72, 96, and 120 h. The florets from each branchlet were then shaved off with a razor blade, frozen in liquid nitrogen and stored at –80 °C.
Arabidopsis thaliana (ecotype Columbia) plants were grown in a controlled environment cabinet (Contherm) under 8/16 h light/dark fluorescent illumination at 23 °C and 60% relative humidity in a 1:1 bark:pumice mix supplemented with 4300 g osmocote plus (15-10-12), 100 g micromax trace elements (Scotts Co., Marysville, OH, USA), 5000 g dolomite, 1000 g superphosphate, 200 g calcium ammonium nitrate, 500 g potassium sulphate, and 100 g terrazole fungicide (Uniroyal Chemical Co., Middlebury, CN, USA) per cubic metre. Plants were sub-irrigated with water as required. Under these non-inductive conditions the plants grew vegetatively for 6 weeks forming a rosette of leaves. Leaves 7 and 8 were used in all analyses and their age was measured in days after leaf emergence (DALE). Zero DALE for leaves 7 and 8 was defined as the time when leaves 5 and 6 were visible (approximately 1 mm in length). Leaves 7 and 8 were harvested from 40 plants (approximately 3-weeks-old), placed on moist tissue and held in the dark at 20 °C. Twenty leaves were removed after 24, 48, 96, and 144 h, frozen in liquid nitrogen and stored at –80 °C. Twenty control leaves were frozen immediately after detachment from the plant. Leaves 7 and 8 were also harvested from 10 plants at 15, 20, 30, and 40 DALE and frozen in liquid nitrogen and stored at 80 °C. Tissue from these leaves was used to access DNA laddering during natural on-plant leaf senescence.
Nucleic acid isolation and analysis
Total RNA and genomic DNA were isolated from broccoli tissue and northerns and genomic Southerns were performed as previously described (Coupe et al., 2003). Total RNA was isolated from Arabidopsis leaves as described previously (Newman et al., 1993). Broccoli and Arabidopsis genomic DNA for DNA laddering experiments was isolated using CTAB following the method described in Current protocols in molecular biology (Ausubel et al., 2001), and Southern analysis was performed as described previously (Eason et al., 2002a).
TUNEL analysis of broccoli
Florets from broccoli branchlets that had been stored for 0, 24, 48, and 72 h after harvest were excised and fixed in a PBS solution (pH 7.4) containing 4% paraformaldehyde and 1% glutaraldehyde (v/v). The fixed tissue was then dehydrated in a graded ethanol series, equilibrated with HistoClearTM II (National Diagnostics) and embedded in paraffin wax (Paraplast, Oxford Labware). Sections (10 µm thickness) of the paraffin-embedded tissue were cut on a retracting microtome (Microm HM340, Heidelberg) and attached to Superfrost® Plus (Erie Scientific, Portsmouth, New Hampshire, USA) microscope slides using standard techniques. DNA strand breaks were detected by direct TUNEL (TdT-mediated dUTP nick end labelling) labelling assays using the in situ cell death detection kit with a fluorescein-dUTP label (Roche, Mannheim, Germany). Nuclear location was determined by counter-staining with DAPI (0.1 mg ml–1 4,6-diamidino-2-phenylindole Molecular Probes). Nuclei were analysed directly under a fluorescence microscope (Olympus BH2-RFCA) using an Olympus UG1 excitation filter, with an Olympus U filter block to detect DAPI, and Olympus B filter to detect fluorescein. The intensity of nuclear fluorescence was determined by video imaging (Video Pro 32 colour image analysis system).
Cloning of putative cell death regulatory genes from broccoli
A 48 h post-harvest broccoli floret cDNA library (Pogson et al., 1995) was screened using heterologous probes from Arabidopsis (LSD1, Dietrich et al., 1997; BI, Sanchez et al., 2000) and potato (STLB1, Birch et al., 1999). Library screening, plasmid DNA isolation and sequencing were carried out as previously described (Coupe et al., 2003).
Arabidopsis PCD regulatory cDNAs
Arabidopsis cDNAs for LSD1 (AtLSD1; U87834
[GenBank]
) and BI (AtBI; AF208124
[GenBank]
) were sourced from overseas laboratories, and SPT (AtSPT1; an EST 163N18T7, accession number R64760
[GenBank]
) was sourced from the Arabidopsis Biological Resource Centre.
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Results |
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Analysis of PCD during senescence using TUNEL (TdT-mediated dUTP nick end labelling)
The nuclei of broccoli sepal cells fluoresced bright green at 24, 48 and 72 h after harvest, indicative of TUNEL fluorescein florescence (Fig. 1). In order to measure the intensity of nuclear staining with fluorescein, images of approximately 100 sepal nuclei were captured at 0, 24, 48, and 72 h and their fluorescence intensity was measured (Fig. 2). The intensity of fluorescein fluorescence of the nuclei population increased from an average of 43 at 0 h to 52 at 24 h, with average intensity levels of 58 and 59 at 48 h and 72 h, respectively. The increase in intensity of fluorescein-stained nuclei is indicative of a greater degree of DNA nicking within the nucleus from 24 h after harvest, a trend that continued as sepal senescence progressed.
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Southern analysis of PCD in broccoli florets and Arabidopsis leaves
DNA was extracted from broccoli florets at 0 h and 1, 6, 24, 48, and 120 h after harvest. DNA was also extracted from non-senescing leaf and stem tissue immediately after harvest as a further control. Southern analysis of the DNA on a membrane that had been probed with 32P-labelled broccoli genomic DNA showed a ladder of DNA fragments at 48 h after detachment (Fig. 3). The DNA ladders (composed of fragments that were multiples of approximately 180 bp in length) were not observed before 48 h and were absent from the leaf and stem samples.
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Two different laddering experiments were conducted with Arabidopsis leaves (Fig. 4). In the first instance the broccoli trial was repeated with detached Arabidopsis leaves to determine whether DNA laddering occurred during this form of harvest-induced senescence in the leaves. DNA was isolated from the 7th and 8th leaves at 21 DALE. DNA ladders (composed of fragments that were multiples of approximately 180 bp in length) were visible at 24, 48 and 96 h after detachment (Fig. 4A). In light of the faint DNA smearing at 0 h for the first experiment (Fig. 4A), leaves were collected at an earlier stage of development (15 DALE) and also after 20, 30 and 40 DALE (at 40 DALE, leaves were 50% chlorotic). DNA laddering was absent in the very young and obviously non-senescing leaves (15 DALE), but was visible from 20 DALE (Fig. 4B); these leaves also did not appear to be yellow and senescing.
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Expression of putative regulatory genes in senescing Arabidopsis leaves
During detached senescence of Arabidopsis leaves, the expression of the Arabidopsis Bax inhibitor (AtBI-1) is high, but remains unchanged throughout the storage period of 0–144 h (Fig. 5). The same trend occurs with AtLSD1, but the constitutive level of expression is much lower than that of AtBI-1, consistent with a low expressing regulatory transcript. AtSPT-1, is not expressed in fresh (0 h) leaves, but accumulates during the senescence period to a maximum level after 144 h of storage (Fig. 5).
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Expression of putative cell death regulatory genes in senescing broccoli florets
Two different LSD1-like cDNAs were isolated from broccoli by heterologous cDNA library screening with Arabidopsis cDNAs. The broccoli cDNAs (BoLSD1 and BoLSD2) were 1027 bp and 1158 bp, respectively, they both had open reading frames of 579 bp and encoded predicted proteins that were 193 amino acids long, with molecular weights of 20.3 and 20.5 kDa, respectively. BoLSD1 and BoLSD2 had long 5' untranslated regions of 158 bp and 214 bp, respectively. The BoLSD1 and BoLSD2 proteins had 84% identity to each other, and 74% and 72% identity with the Arabidopsis LSD1 protein (Fig. 6). A database search with BoLSD1 and BoLSD2 identified other matching sequences previously identified in the Arabidopsis genome that had been given the nomenclature LSD1-like proteins. The Arabidopsis LSD1 homologues have alternate splicing sites that give rise to a low abundance mRNAs encoding an extra five amino terminal amino acids (Fig. 6). Figure 6 also illustrates, with underlining, the presence of three zinc finger domains, defined by CxxCRxxLMYxxGASxVxCxxC (Dietrich et al., 1997), present in the LSD1 class of zinc finger proteins.
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Two putative broccoli BI cDNAs (BoBI-1, BoBI-2) were isolated by heterologous cDNA library screening with the Arabidopsis BI cDNA (AtBI-1, Sanchez et al., 2000). BoBI-1 (1013 bp) encodes a 247 amino acid protein with a predicted molecular weight of 27.5 kDa, whilst BoBI-2 (1019 bp) encodes a 246 amino acid protein with a predicted molecular weight of 27.3 kDa. They share 96% identity with each other and 93% with AtBI-1. The broccoli proteins have identity with several putative BI plant proteins (Fig. 7) with BoBI-1 being 100% identical to a BI protein isolated from B. napus (SA Coupe, unpublished results). In addition, BoBI-1 and BoBI-2 have 42% identity with the first BI protein isolated from humans (Xu and Reed, 1998). Various protein prediction programs have assigned BI proteins either six or seven transmembrane domains (Kawai et al., 1999). This examination reveals that both the BoBI-1 and BoBI-2 proteins are predicted to contain six membrane spanning domains (Fig. 7).
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Two partial cDNAs encoding SPT were isolated from broccoli (BoSPT1 and BoSPT2) by heterologous screening with STLB1 from potato (Birch et al., 1999). BoSPT1 is 603 bp and encodes a predicted protein of 121 amino acids that has 71% identity, in a 259 bp overlap, at the nucleotide level with STLB1. BoSPT1 also had 95% identity with an SPT-like protein that was labelled AtSPT2 (AAK96680 [GenBank] , and 89% identity with the only Arabidopsis SPT characterized to date, AtLCB2 (Tamura et al., 2001). BoSPT1 also has 66% identity at the nucleotide level and 80% identity at the amino acid level with the EST, AtSPT1 (163N18T7, accession number R64760 [GenBank] ), used in earlier northern experiments in the current work. The BoSPT2 sequence was 573 bp and has 72% identity to STLB1 at the nucleotide level, in a 257 bp overlap. BoSPT2 encodes a predicted protein of 103 amino acids and has 94% identity with AAK96680 [GenBank] (SA Coupe, unpublished results) and 88% identity with AtLCB2 (Tamura et al., 2001). BoSPT2 has 67% identity at the nucleotide level and 73% identity at the amino acid level with AtSPT1.
Northern analysis was conducted on total RNA that had been extracted from broccoli floral buds at 0 h and 12, 24, 48, 72, and 96 h after harvest. There was very little BoBI-1 and BoBI-2 transcript in florets at the time of harvest (0 h, Fig. 8A). After 12 h transcripts of approximately 1 kb had accumulated, and for BoBI-1 the transcript levels remained high until 48 h and then fell to negligible levels at 72 h and 96 h. The BoBI-2 transcript accumulated to a maximum level at 48 h and then fell slightly but remained higher than the BoBI-1 levels during the later stages of floret senescence. The 1.1 kb transcripts encoded by BoLSD1 and BoLSD2 were low at 0 h and increased 12 h after harvest, remaining at similar levels as senescence progressed (Fig. 8B). The BoSPT1 and BoSPT2 cDNAs hybridized to transcripts of approximately 2 kb (Fig. 8C). Low levels of transcript were observed at 0 h and 12 h after detachment, after which transcript levels steadily increased for both cDNAs to maximum levels at 96 h (Fig. 8C).
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Genomic Southern analysis was carried out with the full-length cDNAs for BoBI-1, BoBI-2, BoLSD1, and BoLSD2 (Fig. 9). BoLSD1 and BoLSD2 have different hybridization patterns with the digested DNA, but there is some cross-hybridization between the two cDNAs, which share a high sequence identity (84%). The BoBI-1 and BoBI-2 cDNAs have very high sequence identity (96%), and Southern analysis showed BoBI-1 hybridized to one strong band and one fainter band for each restriction digest, whilst BoBI-2 hybridized to the same common single band in each of three different restriction digests (Fig. 9).
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DNA (kb) markers (Invitrogen) have been marked. |
Discussion |
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In this paper it has been shown that substantial DNA processing occurs during detached and natural senescence in plants. Arabidopsis leaves that senesce both naturally on the plant, and after induction by detachment, undergo DNA processing, producing the hallmark ‘ladders’ associated with PCD (Fig. 4). In addition, the senescing floret tissue of harvested broccoli showed DNA laddering within 48 h of harvest (Fig. 3), at a time when the tissues are not visibly deteriorating (sepal yellowing becomes visible 72 h after harvest at 20 °C; Coupe et al., 2003). Using the more sensitive TUNEL assay it was possible to quantify DNA processing associated with PCD in the cells of broccoli floret sepals. Nuclei that had been treated with TUNEL showed greater fluorescence, indicative of DNA nicking, within 24 h of harvest (Figs 1, 2). Previous research in this laboratory has shown that processing of DNA occurs during the senescence of sandersonia flowers (Eason and Bucknell, 2001) and harvest-induced senescence of asparagus spears (Eason et al., 2002a). Other researchers have also shown that DNA processing occurs in senescing Pisum carpels (Orzaez and Granell, 1997) and in pollination-induced petal senescence in petunia petals (Xu and Hanson, 2000). However, the relative timing of this event differs in the different plant systems studied; they occur relatively early in harvested broccoli florets and asparagus spears before external signs of senescence are visible, but relatively late in the senescing floral tissue of sandersonia (tepals) where both fading and wilting precede DNA laddering. It is proposed that the relatively early processing of DNA in harvested broccoli and asparagus relates to the greater stresses imposed on these rapidly growing meristematic tissues by the act of harvest, compared with the programmed senescence of less metabolically active tepals of mature sandersonia flowers.
The expression of three putative regulators of PCD (LSD1, BI, SPT) has been characterized during harvest-induced senescence in Arabidopsis leaves and broccoli florets. LSD1 is a zinc finger protein that functions either negatively to regulate a pro-death pathway component or to activate a repressor of plant cell death (Dietrich et al., 1997). The expression of LSD1 remained constitutively low throughout detached senescence in Arabidopsis leaves. Transcripts for the broccoli LSD1-like genes (BoLSD1, BoLSD2) accumulated between 0 h and 24 h after harvest and then remained constitutively expressed as senescence progressed. The model of LSD1 regulation of PCD (LSD1 has homology to GATA-type transcription factors) does not require up-regulated expression, but rather that expression levels remain constitutive (Dietrich et al., 1997). These results certainly fit within this model.
This is the first isolation of LSD1 homologues from a non-Arabidopsis source, and both broccoli cDNAs have >70% identity with the Arabidopsis protein. The broccoli homologues (BoLSD1, BoLSD2) do not contain the alternative splicing site present in the Arabidopsis genes (Dietrich et al., 1997), and are larger than AtLSD1 with extra proline residues and other sequence differences. They do, however, contain all three zinc finger motifs characteristic of LSD1-like proteins. Southern analysis indicates that there is some cross-hybridization between the two genes, which belong to a small gene family. Recently the EDS1 and PAD4 genes, two signalling genes that mediate some, but not all, plant disease resistance gene responses, have been shown to be essential regulators of the cell death pathway controlled by LSD1 in Arabidopsis (Rusterucci et al., 2001). This discovery provides further tools to investigate the signal transduction pathway operating during PCD in senescence and the similarities between senescence-associated PCD pathways and pathways initiated after pathogen attack.
Although Bax inhibitor homologues have been isolated from rice (Kawai et al., 1999), Arabidopsis (Kawai et al., 1999; Sanchez et al., 2000), barley (Huckelhoven et al., 2001), and now broccoli, and the expressed protein has been shown to suppress Bax-induced cell death in yeast (Kawai et al., 1999; Sanchez et al., 2000; Kawai-Yamada et al., 2001; Yu et al., 2002), the PCD effecter protein, Bax, has not been identified in plants. The significance of plant BI’s, therefore, remains to be determined. The presence of six putative transmembrane domains in the BI proteins (Fig. 7) suggests that the proteins may form ion-conducting channels in a similar manner to the mitochrondria membrane pore-forming Bcl-2 and Bax proteins (Lam et al., 2001). Here, BI expression has been characterized during leaf and floret senescence in Arabidopsis and broccoli, respectively, and it has been shown for the first time that BI is up-regulated during senescence. It has been shown that BI is rapidly up-regulated during wounding and pathogen challenge (Sanchez et al., 2000), and our preliminary results for pathogen-challenged Arabidopsis leaves certainly support these findings (data not shown). In Arabidopsis at least three different BI genes exist (Lam et al., 2001), and two different BI cDNAs have been isolated from broccoli. The broccoli BoBI-1 and BoBI-2 seem to be most similar to AtBI-1, and might encode two isoforms of the AtBI-1 or be two different alleles resulting from F1 broccoli breeding. Southern analysis shows that the two closely related broccoli BI sequences cross-hybridize and are probably encoded by a small gene family. Both BoBI-1 and BoBI-2 transcripts accumulate rapidly during harvest-induced senescence before any significant DNA processing, which occurs from 48 h after harvest. This indicates that BI may be playing a significant role in the PCD signalling that occurs during detached senescence in broccoli. The challenge is to demonstrate that BI does form a membrane channel and to determine its occurrence in the cell and the other proteins that interact with it.
Ceramide is thought to act as a regulatory molecule during apoptosis. Human studies have shown that the de novo pathway of sphingolipid synthesis is a novel way of generating ceramide during apoptosis (Perry et al., 2000). In addition, sphingolipids and their associated metabolites (e.g. ceramide, sphingosine and sphingosine-1-phosphate) are thought to be involved in the regulation of a diverse range of processes in plants, including pathogenesis, membrane stability and the response to drought (Ng et al., 2001). One of the first homologues of SPT (the enzyme required for the formation of the C18 long-chain base sphinganine; Lynch and Fairfield, 1993) isolated from plants was found in potato tubers that were undergoing PCD during the HR (Birch et al., 1999). In the current study, it has been shown that, in Arabidopsis, SPT transcripts accumulate during leaf senescence. During harvest-induced senescence of broccoli florets, the transcripts of broccoli SPT homologues (BoSPT1, BoSPT2) accumulated to maximum levels at 96 h, mirroring the senescence-associated increase in AtSPT seen in detached, senescing Arabidopsis leaves. Sphinglolipid metabolites formed by the activity of SPT are thought to be involved in the signalling that accompanies the stress of detachment or pathogen infection and thereby regulate the subsequent PCD processes that occur (Ng et al., 2001). Indeed, in a recent publication, the isolation and characterization of an Arabidopsis accelerated-cell-death mutant (acd11), revealed that the ACD11 gene encodes a sphingosine transfer protein (Brodersen et al., 2002). The knockout mutant has an accelerated PCD and defence response.
In conclusion, broccoli florets express similar genes during detached (harvest-induced) senescence to those expressed in Arabidopsis leaves during senescence. During detached senescence and natural on-plant senescence DNA is degraded into fragments; multiples of approximately 180 bp in length. This pattern of DNA laddering is one of the hallmarks of PCD in animal cells and, more recently, has also been identified as an element in certain PCD pathways in plants (reviewed in Plant Molecular Biology, Vol. 44). It has been shown here that the expression of broccoli and Arabidopsis homologues of LSD1, BI and SPT is associated with PCD during senescence. In particular, the broccoli LSD1 and BI homologues are up-regulated at 12 h and 24 h, prior to DNA degradation and before the onset of visible senescence (72 h). This is also the time when cysteine protease transcription increases (Coupe et al., 2003). The relatively early timing of DNA fragmentation in harvested broccoli is related to the greater stresses imposed on the actively growing meristematic tissues through the act of harvest.
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Acknowledgements |
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The authors wish to thank Murray Grant (Imperial College, London Wye campus, Kent, UK) for supplying the AtBI-1 cDNA; Jeff Dangl (University of North Carolina, Chapel Hill, NC, USA) for supplying the LSD1 cDNA; Paul Birch (SCRI, Invergowrie. UK) for supplying STLB1; and the ABRC (Columbus, OH, USA) for supplying the AtSPT1 EST. The authors also wish to thank local broccoli grower, Ormond Curry, for supplying plant material; Ian King for the growth and maintenance of Arabidopsis plants, and the NZ Foundation for Research, Science and Technology for funding.
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References |
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