JXB Advance Access originally published online on March 12, 2004
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Journal of Experimental Botany, Vol. 55, No. 398, pp. 889-897, April 1, 2004
© 2004 Oxford University Press
Regulation of Growth, Development and Whole Organism Physiology |
Caspase-like activity in the seedlings of Pisum sativum eliminates weaker shoots during early vegetative development by induction of cell death
Received 19 June 2003; Accepted 24 December 2003
Department of Plant Sciences, The Hebrew University of Jerusalem, Givat-Ram Campus, Jerusalem 91904, Israel
* To whom correspondence should be addressed. Fax: +972 2 658 4425. E-mail:alexl{at}cc.huji.ac.il
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Abstract |
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Activation of aspartate-specific cysteine proteases (caspases) plays a crucial role in programmed cell death (PCD) in animals. Although to date caspases have not been identified in plants, caspase-like activity was described in tobacco during a hypersensitive response to pathogens and in Arabidopsis and tomato cell cultures during chemical-induced PCD. Caspase-like activity was also detected in the course of plant development during petal senescence and endosperm PCD. It is shown here that caspase-like proteases play a crucial role in the developmental cell death of secondary shoots of pea seedlings that emerge after removal of the epicotyl. Caspase-like activity was induced in senescing secondary shoots, but not in dominant growing shoots, in contrast to the papain-like cysteine protease activity that was stronger in the dominant shoot. Revitalization of the senescing shoot by cutting of the dominant shoot reduced the caspase-like activity. Injection of caspase or cysteine protease inhibitors into the remaining epicotyl tissue suppressed the death of the secondary shoots, producing seedlings with two equal shoots. These results suggest that shoot selection in pea seedlings is controlled by PCD, through the activation of caspase-like proteases.
Key words: Apical dominance, cysteine proteases, epicotyl, senescence, shoot development.
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Introduction |
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Pea seedlings constitute the classical model for studying the process of shoot selection in early development of the apical meristem. Original work by Snow showed that when a 7-d-old pea epicotyl is cut off, two new shoots develop below the cut surface. Initially, they are of equal size, but one of them soon establishes apical dominance by a presently unknown mechanism, while the secondary shoot stops growing and rapidly ages (Snow, 1931, 1932). The apical dominance of the more robust shoot represents an extreme case of dominance, which invariably results in the death of the secondary shoot. However, if the dominant shoot is removed before death occurs, then the weak shoot is revitalized and starts growing. Importantly, it was shown that the death of the weak shoot does not depend on endogenous lack of nutrients, suggesting an active process regulated by internal stimuli (Sachs, 1966). This process, particularly its initiation, is subject to regulation by many environmental and autonomous (internal) factors. In general, conditions that are unfavourable for growth accelerate the selection of the dominant shoot and the senescence and cell death of the secondary shoot.
Senescence is a final phase of plant vegetative and reproductive development, preceding the widespread death of cells and organs. Leaf senescence is the final stage of leaf development. Physiological, biochemical, and molecular studies show that, during senescence, leaves undergo highly co-ordinated changes in cell structure, metabolism, and gene expression (Gan and Amasino, 1997). The requirements for new RNA and protein synthesis imply that leaf senescence is a genetically programmed cell death (PCD) process (Crafts Brandner et al., 1996). Leaf senescence can be considered as the archetype of PCD, and, in fact, it has been shown to exhibit the hallmarks of apoptotic cell death (Greenberg, 1996; Caccia et al., 2001). PCD or apoptosis significantly differ from necrotic cell death in the biochemical mechanism and in the subsequent biological effects, particularly in pathological implications (Steller, 1995; Beers, 1997).
The earliest and most significant change in leaf cell structure is the breakdown of the chloroplast, the organelle that contains up to 70% of the leaf protein. During this process carbon assimilation (photosynthesis) is replaced by catabolism of chlorophyll and other macromolecules such as proteins, lipids, and RNA. Senescence is a tightly regulated developmental process that involves turnover and recapture of cellular material, ultimately resulting in cell death (Beers, 1997; Noodén et al., 1997). It is influenced by many hormones, environmental conditions, and signalling molecules (Quirino et al., 2000).
Apoptosis, which is a highly regulated form of PCD, is a major form of cell death in animals, plants, and even in yeast (Raff et al., 1994; Pennell and Lamb, 1997; Frohlich and Madeo, 2000). In animals, PCD is characterized by several morphological and biochemical hallmarks, such as shrinkage of the cytoplasm, nuclear condensation, membrane blebbing, and induction of proteases and endonucleases (Steller, 1995). While these processes have been also observed in plant PCD, they are not associated with every type of PCD in plants (Pennell and Lamb, 1997). Frequently, only some of the hallmarks accompany plant PCD, suggesting the existence of several alternative cell death pathways (Greenberg, 1996). Variations in the mechanisms of PCD were also described in animal systems.
Molecular studies in nematodes and animals have found that apoptosis is controlled by a proteolytic cascade that involves a set of conserved cysteine proteases, caspases, which cut target proteins after an aspartate residue (Thornberry and Lazebnik, 1998). Cysteine proteases also play a crucial role in many forms of plant PCD, including senescence (Solomon et al., 1999; Heath, 2000). The induction and the function of true caspase-like activities in plants has been recently reviewed in Woltering et al. (2002). The predominant class of senescence-induced proteases belongs to the cysteine protease family (Valpuesta et al., 1995; Drake et al., 1996; Ye and Varner, 1996). In castor beans, papain-like cysteine protease was shown to accumulate in senescing plant tissue, concomitant with the appearance of DNA cleavage, indicating the induction of PCD (Schmid et al., 2001). Moreover, inhibition of the cysteine protease activity by chemical inhibitors suppressed the leaf senescence process (Beers, 1997; Noodén et al., 1997).
The high specificity of caspases towards their substrates makes them ideal enzymes for the regulation of crucial decisions in the cell. Indeed, the caspase-dependent pathway is the major type of apoptosis in many animal systems, although a caspase-independent pathway was also described (Cohen, 1997). Although caspase homologues were not found in plants, the complete sequence of the Arabidopsis genome revealed the presence of several metacaspase genes (Uren et al., 2000). Recently, caspase-like activity was detected in protein extracts from tobacco plants during a hypersensitive reaction, which is regarded as a form of PCD (del Pozo and Lam, 1998). Caspase-like activity was also detected in Arabidopsis and tomato cell cultures undergoing PCD triggered by nitric oxide or by other chemicals that also induce PCD in animals (Clarke et al., 2000; De Jong et al., 2000). Specific peptide inhibitors of caspases suppressed camptothecin-induced PCD in tomato suspension cultures, supporting possible caspase(s) activity in plants (Orzaez et al., 2001). Moreover, tomato plants that expressed the antiapoptotic baculovirus p35 gene, which interacts with the active site of the target caspases, blocked PCD (Lincoln et al., 2002).
It is shown here that the induced senescence and cell death of the secondary shoots in pea seedlings is regulated by a caspase-like activity, which occurs concurrently with increased oxidation. Inhibition of the caspase-like activity affected the natural morphogenesis after epicotyl removal, and resulted in the growth of plants with two equal-sized shoots.
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Materials and methods |
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Chemicals
Protease substrates were from Calbiochem-Novabiochem (La Jolla, CA, USA). Other chemicals were from Sigma (St Louis, MO, USA). Peptides were dissolved in DMSO and diluted in water before addition to the reaction mix or injection into seedlings. Amastatin was dissolved in ethanol.
Plant material
Pea seeds (Pisum sativum var. arvense Poir. cv. Dun) were sown in vermiculite. The plants were grown at 21 °C and 12 h of light (275 µmol m–2 s–1 light intensity). One week after germination, plants were transferred into hydroponic conditions with Hoagland solution, and the seminal shoots were cut off. Two new shoots developed within 7 d, one of which became dominant and the other senesced and started to die unless the dominant shoot was removed.
Protease assays
Proteins were extracted from shoots frozen in liquid nitrogen by grinding 1 g of tissue in a cold mortar and then adding 5 ml of 200 mM phosphate buffer pH 7, 1 mM EDTA, and 1% polyvinylpolypyrrolidone. Samples were centrifuged for 10 min at 14 000 g, 4 °C and the supernatant was used for the protease assay. Protein concentration was determined according to Bradford (1976), and 20 µg of proteins were used for the protease activity assay. The reactions were adjusted to 100 µl volume in buffer (20 mM MES pH 6.6, 0.25 mM dithiothreitol (DTT), 100 mM NaCl, 2.5 mM EDTA, and 2.5 µg ml–1 fluorogenic substrate). The reactions with inhibitors were incubated for 20 min at 30 °C prior to addition of the substrates.
Proteolytic activity was measured in a FL600 fluorometer (BioTek, VI, USA; excitation 360/40; emission 460/40, sensitivity 100). The blank fluorescence readings (minus substrate) were subtracted. The following fluorogenic substrates were used: t- benzyloxycarbonyl (CBZ)-Gly-Gly-Arg-AMC (GGA) and N
- benzyloxycarbonyl (CBZ)-Arg-Arg-AMC (AA) for testing general cysteine protease activities; t-butyl-oxycarbonyl (BOC)-Gly-Lys-Arg-AMC (GLA) for analysis of the trypsin activity; caspase-like activity was assayed with 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL)-Tyr-Val-Ala-Asp-Ala-Pro-Val-5-([2-aminoethyl]amino)naphthalene-1-sulphonic acid (EDANS) and with Ac-YVAD-AMC substrates. All substrates were dissolved in DMSO and diluted in water to the final concentration. Caspase inhibitor (Ac-Asp-Glu-Val-Asp-CHO) was used at 0.2 mM or 20 nM concentrations [which were found to be effective in plants (Woltering et al., 2002)] for in vitro (see Figs 4 and 5 in the Results) and in vivo (see Fig. 6 and Table 1 in the Results) experiments, respectively. The general cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) was used at a 10 µM concentration throughout this study.
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In gel protease assay
Proteins (20 µg) were incubated for 5 min at 30 °C in sample buffer and separated in a 10% SDS-polyacrylamide gel. The gel was washed twice with 50 mM TRIS, pH 7.5, and 0.25% Triton X-100 for 45 min each and renatured overnight at 30 °C in 10 mM TRIS, pH 7.5, and 0.25% Triton X-100. Activity was detected in a solution of 20 mM MES pH 6.6, 0.25 mM dithiothreitol (DTT), 100 mM NaCl, 2.5 mM EDTA, and 2.5 µg ml–1 Ac-YVAD-AMC substrate. The fluorescence in the gel was followed in real time by impregnating the ICE substrate into the gel and viewing with an ImageMaster VDS system (Pharmacia) equipped with a 360 nm excitation lamp above the gel and a 460 nm emission filter.
Lipid peroxidation
Lipid hydroperoxides were measured with a Lipid Hydroperoxide (LPO) Assay Kit (Cayman Chemicals, USA), according to the manufacturer’s instructions. The total cell extract from the pea shoot was normalized according to the protein concentration. Equal amounts of extraction buffer, saturated in methanol, were added and the samples were vortexed for 2 min. One ml of cold chloroform was added and the samples vortexed again. After centrifugation for 5 min at 1500 g at 4 °C, the bottom chloroform layer was collected and mixed with 450 µl of chloroform:methanol 2:1 (v:v) mix and 50 µl of chromogen (as supplied in the kit). The reactions were done in 300 µl by adjusting the volume with water. The absorbance was read against chloroform:methanol (without chromogen) at 500 nm and compared with a standard curve.
Northern blot analyses
Total RNA was extracted from the pea shoot at different times using the RNeasy Plant Mini Kit (QIAGEN) as described by the manufacturer. Five micrograms of total RNA were separated on 1.5% (w/v) agarose denaturing formaldehyde gels, and RNA was visualized by staining the gels with ethidium bromide (1 mg ml–1). The separated RNAs were subsequently blotted onto Hybond N+ membrane (Amersham Pharmacia Biotech), according to the manufacturer’s instructions, and cross-linked by UV irradiation. The Arabidopsis SAG12 was used as a probe and radioactive probe. Hybridization was performed as previously described (Govrin and Levine, 2000).
Chromatography
Cleared protein extracts were loaded onto a Biologic (Bio-Rad) medium pressure chromatography system with an anion exchange column of 1 ml UNO-Q. Proteins were eluted with a linear NaCl gradient at 1 ml min–1.
ROS measurements
The amount of ROS in shoots of pea plants was measured according to (Lu and Higgins (1998). Briefly: the samples (at least in triplicate) were washed for 90 min in water with vigorous shaking, and then placed into the reaction buffer (10 mM TRIS pH 8, 10 units horseradish peroxidase, 10 µM 2',7'-dichlorofluorescein diacetate) in a final volume of 200 µl. The samples were kept in the dark for 10 min and read using a BioTek FL600 fluorometer with the following conditions: excitation 485/40; emission 530/40; sensitivity 100. The fluorescent probe was prepared by diluting 2 mg ml–1 ethanol stock solution. A standard curve was prepared before each experiment using different H2O2 concentrations in fresh medium. The measure was normalized according to the dry weight.
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Results |
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Developmental and physiological changes in establishment of shoot dominance
To study the process of shoot selection in early development of pea seedlings the apical meristems were cut off 7 d after germination. Two new shoots emerged below the cut. The growth of the shoots was disproportionate, resulting in a larger leading branch and a small secondary one (Fig. 1A). The secondary shoot died within 21 d when left untouched. However, cutting off the dominant shoot stopped the rapid senescence of the secondary shoot, and re-established its growth rate, indicating that the process was reversible (Fig. 1B).
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To see whether the deterioration of the secondary shoot was associated with the expression of senescence-associated genes from other systems, the RNA from each shoot was probed with a genetic marker of the SAG12 gene that was shown to be completely senescence-specific. This gene was first isolated from naturally senescencing Arabidopsis leaves (Lohman et al., 1994), but homologous genes were found in other plants as well (Ori et al., 1999; Pontier et al., 1999). Northern analysis of SAG12 gene expression showed that it was present predominantly in the dying branch. Interestingly, its transcription stopped after cutting off the dominant shoot, indicating that the SAG12 gene expression depended on the continuation of the senescence programme (Fig. 2).
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In some species leaf senescence is accompanied by a respiratory burst, resulting in development of an oxidative stress (Casano et al., 1994; Merzlyak and Hendry, 1994; Heber et al., 1996). Production of reactive oxygen species (ROS) during the senescence of the dying (secondary) branch was examined by measuring oxidation of 2',7'-dichlorofluorescein diacetate (DCFH-DA) in the dominant and the secondary shoots (Lee et al., 1999; Murata et al., 2001). Shoot cuttings were incubated in DCFH-DA solution for 10 min in the dark and the amount of oxidized dye measured by spectrofluorophotometry. The dying shoot produced consistently more ROS (Fig. 3A). The increase in ROS production was detected 11 d after removal of the apical meristem, coinciding with the beginning of dominance establishment. If not counteracted by a corresponding increase in antioxidant activity, oxidative stress leads to increased lipid peroxidation. More than 3-fold higher levels of lipid hydroperoxides were detected in the dying shoot, in line with increased ROS in that branch (Fig. 3B).
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Proteolytic activity during induction and reversal of senescence
Caspase induction is a central step in animal PCD, including the ageing-associated cell death (Wang et al., 1998). Recently, caspase-like activity was also discovered in plants. Protease involvement in shoot death was analysed 1 week after cutting off the primary bud by assaying the proteolytic activity in the dominant and secondary shoot extracts by measuring the cleavage of several fluorogenic peptide substrates. Cysteine proteases were assayed by using two different substrates, t-benzyloxycarbonyl (CBZ)-Gly-Gly-Arg-AMC (GGA) and N
-benzyloxycarbonyl (CBZ)-Arg-Arg-AMC (AA). Trypsin-like activity was assayed with t-butyl-oxycarbonyl (BOC)-Gly-Lys-Arg-AMC (GLA), which is cut preferentially by trypsin (Wilson et al., 2002). Caspase-like activity was measured with a relatively long substrate, 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL)-Tyr-Val-Ala-Asp-Ala-Pro-Val-5-([2-aminoethyl]amino) naphthalene-1-sulphonic acid (EDANS), that is specific for interleukin-1 beta converting enzyme (ICE) as well as the ICE homologue apopain/CPP-32/Yama (Pennington and Thornberry, 1994; Hugunin et al., 1996). Proteolytic activity using the two broad-range cysteine protease substrates or the trypsin-type peptide revealed a rather similar pattern in the short and in the long shoots (Fig. 4A). Furthermore, their activities were not significantly altered after cutting off the leading shoot. By contrast, the caspase-specific substrate showed a strong activity only in the short secondary shoot, suggesting the induction of a senescence-associated protease (Fig. 4A). Inclusion of an aminopeptidase inhibitor, amastatin (10 µM), did not alter the activity, indicating that the fluorescent signal was dependent on endopeptidase activity (data not shown). Exclusive activity in the dying secondary shoot was also observed with short caspase-specific peptide substrates, benzyloxycarbonyl (Cbz) Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin (YVAD) or Ac-Asp-Glu-Val-Asp-AMC (DEVD) that are preferentially cleaved by caspases 1 and 3, respectively, although it should be mentioned that YVAD also acts as a substrate for caspases 3, 4, and 7 (Villa et al., 1997) (data not shown). Importantly, the proteolysis of the ICE substrate was suppressed by very low concentrations (0.2–0.5 nM) of reversible caspase inhibitors (Ac-Asp-Glu-Val-Asp-CHO or Ac-Tyr-Val-Ala-Asp-CHO). A relatively high dosage of 10 µM of a general cysteine protease inhibitor, E-64, inhibited both the aspartate- and the arginine-specific proteases. Taken together, the substrate specificity and the inhibition profile of the proteases support the involvement of a cysteine protease with caspase-like target specificity.
The time-course of caspase-like activity was studied in protein extracts from the leading and the senescing shoots, beginning at 3 d after cutting of the epicotyl (which was the earliest time that enough biological material could be collected for protein analysis in the small shoot). Proteins were concentrated and assayed with the long ICE substrate. Increased caspase-like activity was already detected in the short shoot 3 d after cutting, and it remained high throughout the duration of the experiment (Fig. 4B). By contrast, when the same samples were assayed with the general substrate of cysteine proteases (GGA), stronger activity was observed in samples from the long shoot, indicating that the increased proteolysis of the caspase substrate in the dying shoot was not due to a general up-regulation of proteases (Fig. 4C).
Partial purification of the caspase protein from the dying secondary shoot
To characterize the caspase-like activity, total protein extracts were prepared from the senescing shoot. The caspase-like proteolytic activity was recovered by precipitation with 30% ammonium sulphate. The precipitate was dialysed and loaded onto a cation exchange column and eluted with a NaCl gradient. The caspase-like activity that appeared in the flowthrough (data not shown) was loaded on an anion exchange column. A clear difference was seen in the profile of proteins eluted with a linear gradient of NaCl, indicating major changes in protein expression during shoot senescence (Fig. 5A). Each fraction was analysed by testing the activity with the long ICE substrate. The proteolytic activity was only recovered in two adjacent fractions towards the end of the NaCl gradient, between 0.6–0.7 µS, indicating the acidic nature of the protease (Fig. 5B).
Proteolytic activity was also detected in these fractions using the short caspase-1 (Ac-YVAD-AMC) or caspase-3 (Ac-DEVD-AMC)-specific substrates (data not shown). To obtain information on the molecular size of the caspase-like protease, the eluted fractions were concentrated and analysed in a polyacrylamide gel following SDS-PAGE. Proteolytic activity was assayed by impregnation of the short fluorogenic substrate (Ac-YVAD-AMC), which acts as a substrate for caspases 3, 4, and 7. The activity was visualized in an ImageMaster VDS system (Pharmacia). The active bands corresponded to apparent molecular weight of 55 kDa (Fig. 5C).
Inhibition of caspases leads to morphological changes in seedling development
The above results implied a possible involvement of caspase-like proteins in the death of the secondary shoot. In order to test whether caspases play a shaping role in vivo in the death of the shoot, various protease inhibitors were injected into the stem of the epicotyl, immediately after the removal of the apical meristem. The subsequent additions were done by direct injections into the secondary shoot. Application of the inhibitors with anti-cysteine protease activity reversed the death process in the secondary branch. Injection of the caspase-3 inhibitor strongly inhibited the death of the secondary branch, resulting in the almost equal growth of both shoots (Table 1). A slightly lower protection was also observed by the general cysteine protease inhibitor, E-64, and by the serine-cysteine protease inhibitor, AEBSF (Fig. 6). It should be noted that AEBSF was shown to inhibit cysteine proteases in plants and hypoxia-reoxygenation-induced apoptosis in rat kidney cells by the inhibition of caspase-9 activity (Dong et al., 2000). By contrast, the addition of serine protease inhibitors (25 µM alanyl-alanyl-phenylalanyl-chloromethylketone or 2 µM aprotinin) had no effect on secondary shoot development (Table 1).
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Discussion |
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Apical dominance is defined as the inhibition of lateral buds by the dominant apex. The inhibition may be partial, causing slower development of the secondary branches or alteration of their growth direction, or it can completely stop their growth. Pea plants exhibit an extreme case of apical dominance, whereby the weaker shoot is actually killed (Snow, 1931). Interestingly, removal of the epicotyl causes both branches to grow equally well. However, once one of the shoots gains advantage due to some stochastic event, such as shading, pathogen attack, or insect-caused damage the other shoot rapidly establishes dominance and induces death of the weaker branch. The death process can be rapidly and completely reversed by removing the dominant cotyledonary bud (Fig. 1). The activation of different proteases and the reversibility of the death process suggests an active cell death process that is associated with senescence.
In animal systems, caspase activation has emerged as a key step in the regulation of developmental apoptosis (Martin and Green, 1995; Xue and Horvitz, 1995). Although non-caspase-dependent PCD also operates in animals, caspases are critical for developmentally induced apoptosis (Cohen, 1997). For example, in nematodes, mutations in the caspase gene, Ced-3, prevent cell death of specific cells, while ectopic expression of caspase genes causes additional cell death (Xue and Horvitz, 1995). In plants, developmentally induced PCD occurs in aleurone and in unpollinated ovules, during aerenchyma and formation of tracheary elements. In some plants PCD causes leaf lobes and perforations (Pennell and Lamb, 1997). To date none of these processes has been associated with caspases or caspase-like activity. However, cysteine proteases were shown to be activated during xylogenesis and in flower and leaf senescence (Minami and Fukuda, 1995).
On the other hand, caspase-like activity was reported for several stress-triggered PCD processes, such as hypersensitive reaction and chemical treatments (del Pozo and Lam, 1998; Sun et al., 1999; De Jong et al., 2000). The involvement of caspase-like activity for execution of the PCD in plants is also supported by inhibitor studies, whereby addition of either caspase-3 inhibitor (Ac-DEVD-CHO) or caspase-1 inhibitor (Ac-YVAD-CMK) suppressed hypersensitive cell death from avirulent bacteria infection or from mycotoxin induced PCD (Woltering et al., 2002). The caspase-specific inhibitors also effectively blocked PCD from chemical and heat shock treatments (Sun et al., 1999; De Jong et al., 2000). These results show that caspase-like activity operates in developmental PCD in pea seedlings, that is associated with organ senescence.
The process of senescence is tightly associated with oxidative stress (del Rio et al., 1992; Smirnoff, 1993; Pell et al., 1997). Increased H2O2 concentration and lipid peroxidation occur in leaves during senescence (Jimenez et al., 1998; Berger et al., 2001). ROS also play a major role in PCD induction in plants (Levine et al., 1994; Noctor and Foyer, 1998; Mazel and Levine, 2001). Treatment of soybean cells with H2O2 induced cysteine proteases, which were instrumental in their PCD (Solomon et al., 1999; Heath, 2000). In Arabidopsis cells, H2O2-triggered PCD could be suppressed by cysteine protease inhibitors including caspase-specific inhibitors (Tiwari et al., 2002). In the present study, ROS production in the dying shoot was correlated with protease activation, suggesting that ROS may be involved in the signalling of PCD in the secondary branch (Fig. 3).
Several factors point to the possible involvement of caspase-like proteases in the death of the weaker secondary shoot. The proteolytic caspase-like activity was only induced in the dying shoot (Fig. 4). Importantly, the papain-like activity, on the contrary, was even slightly stronger in the dominant shoot. Moreover, the molecular weight of the caspase-specific activity (Fig. 5C) was much larger than that of the papain-like cysteine proteases. Judging by the molecular weight of the proteolytic activity, the pea protein may be similar to human initiator caspases-8 and -10. However, direct comparison will only be possible after cloning of the pea protease. The caspase-like activity was inhibited by subnanomolar concentrations of a caspase inhibitor. Inhibition of the protease activity completely blocked the death process and produced plants with equal shoots, indicating that the control exerted by the dominant shoot is mediated via a caspase-like protease (Fig. 6; Table 1).
To date the caspase-like activity in plants has been detected only in cases associated with either severe stresses, such as heat shock (Tian et al., 2000), treatment with toxic chemicals (Sun et al., 1999; De Jong et al., 2000), pathogen infection (del Pozo and Lam, 1998), or with specific developmental events, such as petal senescence (Xu and Hanson, 2000) or endosperm cell death during germination (Schmid et al., 1999). A relatively broad range of caspase-specific substrates has been observed in the present study and in the other plant systems that have been analysed with several substrates (Woltering et al., 2002). This could be a result of a simultaneous induction of several different caspase-like proteases, or alternatively, it could stem from a relaxed stringency in substrate recognition in plants. Both these phenomena have been described in the well characterized animal systems (Cohen, 1997; Margolin et al., 1997; Rano et al., 1997; Bae et al., 2001).
In summary, it has been shown that protease(s) with caspase-like activity play a determinative role in the early vegetative development of pea seedlings. Since the sequencing projects of model plant genomes have not yet identified true caspases, it is difficult to attribute the cleavage of the various caspase-specific substrates to specific enzymes. Since the pea genome is not expected to be sequenced soon, the isolation of the caspase-like protease is necesary for analysing the substrate specificity. Unfortunately, the activity is present only in the dying shoots, which constitutes a very small amount of available biological material for purification.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Bae SS, Choi JH, Oh YS, Perry DK, Ryu SH, Suh PG. 2001. Proteolytic cleavage of epidermal growth factor receptor by caspases. FEBS Letters 491, 16–20.[CrossRef][Web of Science][Medline]
Beers EP. 1997. Programmed cell death during plant growth and development. Cell Death and Differentiation 4, 649–661.
Berger S, Weichert H, Porzel A, Wasternack C, Kuhn H, Feussner I. 2001. Enzymatic and non-enzymatic lipid peroxidation in leaf development. Biochimica et Biophysica Acta—Molecular and Cell Biology of Lipids 1533, 266–276.[CrossRef]
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254.[CrossRef][Web of Science][Medline]
Caccia R, Delledonne M, Levine A, De Pace C, Mazzucato A. 2001. Apoptosis-like DNA fragmentation in leaves and floral organs preceeds their developmental senescence. Plant Biosystems 135, 183–190.
Casano LM, Martin M, Sabater B. 1994. Sensitivity of super oxide-dismutase transcript levels and activities to oxidative stress is lower in mature-senescent than in young barley leaves. Plant Physiology 106, 1033–1039.[Abstract]
Clarke A, Desikan R, Hurst RD, Hancock JT, Neill SJ. 2000. NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures. The Plant Journal 24, 667–677.[CrossRef][Web of Science][Medline]
Cohen GM. 1997. Caspases: The executioners of apoptosis. Biochemistry Journal 326, 1–16.
Crafts Brandner SJ, Klein RR, Klein P, Hoelzer R, Feller U. 1996. Coordination of protein and mRNA abundances of stromal enzymes and mRNA abundances of the Clp protease subunits during senescence of Phaseolus vulgaris L.) leaves. Planta 200, 312–318.[Web of Science][Medline]
De Jong AJ, Hoeberichts FA, Yakimova ET, Maximova E, Woltering EJ. 2000. Chemical-induced apoptotic cell death in tomato cells: involvement of caspase-like proteases. Planta 211, 656–662.[CrossRef][Web of Science][Medline]
del Pozo O, Lam E. 1998. Caspases and programmed cell death in the hypersensitive response of plants to pathogens. Current Biology 8, 1129–1132.[CrossRef][Web of Science][Medline]
del Rio LA, Sandalio LM, Palma JM, Bueno P, Corpas FJ. 1992. Metabolism of oxygen radicals in peroxisomes and cellular implications. Free Radical Biology in Medicine 13, 557–580.
Dong Z, Saikumar P, Patel Y, Weinberg JM, Venkatachalam MA. 2000. Serine protease inhibitors suppress cytochrome c-mediated caspase-9 activation and apoptosis during hypoxia-reoxygenation. Biochemistry Journal 347, 669–677.
Drake R, John I, Farrell A, Cooper W, Schuch W, Grierson D. 1996. Isolation and analysis of cDNAs encoding tomato cysteine proteases expressed during leaf senescence. Plant Molecular Biology 30, 755–767.[CrossRef][Web of Science][Medline]
Frohlich KU, Madeo F. 2000. Apoptosis in yeast – a monocellular organism exhibits altruistic behaviour. FEBS Letters 473, 6–9.[CrossRef][Web of Science][Medline]
Gan S, Amasino RM. 1997. Making sense of senescence: molecular genetic regulation and manipulation of leaf senescence. Plant Physiology 113, 313–319.[CrossRef][Web of Science][Medline]
Govrin EM, Levine A. 2000. The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea. Current Biology 10, 751–757.[CrossRef][Web of Science][Medline]
Greenberg JT. 1996. Programmed cell death: a way of life for plants. Proceedings of the National Academy of Sciences, USA 93, 12094–12097.
Heath MC. 2000. Hypersensitive response-related death. Plant Molecular Biology 44, 321–334.[CrossRef][Web of Science][Medline]
Heber U, Miyake C, Mano J, Ohno C, Asada K. 1996. Mono dehydroascorbate radical detected by electron paramagnetic resonance spectrometry is a sensitive probe of oxidative stress in intact leaves. Plant and Cell Physiology 37, 1066–1072.
Hugunin M, Quintal LJ, Mankovich JA, Ghayur T. 1996. Protease activity of in vitro transcribed and translated Caenorhabditis elegans cell death gene (ced-3) product. Journal of Biological Chemistry 271, 3517–3522.
Jimenez A, Hernandez JA, Pastori G, del Rio LA, Sevilla F. 1998. Role of the ascorbate–glutathione cycle of mitochondria and peroxisomes in the senescence of pea leaves. Plant Physiology 118, 1327–1335.
Lee S, Choi H, Suh S, Doo I-S, Oh K-Y, Jeong Choi E, Schroeder Taylor AT, Low PS, Lee Y. 1999. Oligogalacturonic acid and chitosan reduce stomatal aperture by inducing the evolution of reactive oxygen species from guard cells of tomato and Commelina communis. Plant Physiology 121, 147–152.
Levine A, Tenhaken R, Dixon R, Lamb C. 1994. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79, 583–593.[CrossRef][Web of Science][Medline]
Lincoln JE, Richael C, Overduin B, Smith K, Bostock R, Gilchrist DG. 2002. Expression of the antiapoptotic baculovirus p35 gene in tomato blocks programmed cell death and provides broad-spectrum resistance to disease. Proceedings of the National Academy of Sciences, USA 99, 15217–15221.
Lohman KN, Gan SS, John MC, Amasino RM. 1994. Molecular analysis of natural leaf senescence in Arabidopsis thaliana. Physiologia Plantarum 92, 322–328.[CrossRef]
Lu H, Higgins VJ. 1998. Measurement of active oxygen species generated in planta in response to elicitor AVR9 of Cladosporium fulvum. Physiology and Molecular Plant Pathology 52, 35–51.
Margolin N, Raybuck SA, Wilson KP, Chen W, Fox T, Gu Y, Livingston DJ. 1997. Substrate and inhibitor specificity of interleukin-1-beta-converting enzyme and related caspases. Journal of Biological Chemistry 272, 7223–7228.
Martin SJ, Green DR. 1995. Protease activation during apoptosis: death by a thousand cuts? Cell 82, 349–352.[CrossRef][Web of Science][Medline]
Mazel A, Levine A. 2001. Induction of cell death in arabidopsis by superoxide in combination with salicylic acid or with protein synthesis inhibitors. Free Radical Biology in Medicine 30, 98–106.[CrossRef]
Merzlyak MN, Hendry GAF. 1994. Free radical metabolism, pigment degradation and lipid peroxidation in leaves during senescence. Proceedings of the Royal Society of Edinburgh 102, 459–471.
Minami A, Fukuda H. 1995. Transient and specific expression of a cysteine endopeptidase associated with autolysis during differentiation of Zinnia mesophyll cells into tracheary elements. Plant Cell Physiology 36, 1599–1606.
Murata Y, Pei ZM, Mori IC, Schroeder J. 2001. Abscisic acid activation of plasma membrane Ca2+ channels in guard cells requires cytosolic NAD(P)H and is differentially disrupted upstream and downstream of reactive oxygen species production in abi1-1 and abi2-1 protein phosphatase 2C mutants. The Plant Cell 13, 2513–2523.
Noctor G, Foyer CH. 1998. Ascorbate and glutathione: keeping active oxygen under control. Annual Review of Plant Physiology and Plant Molecular Biology 49, 249–279.[CrossRef][Web of Science]
Noodén LD, Guiamet JJ, John I. 1997. Senescence mechanisms. Physiologia Plantarum 101, 746–753.[CrossRef]
Orzaez D, de Jong AJ, Woltering EJ. 2001. A tomato homologue of the human protein PIRIN is induced during programmed cell death. Plant Molecular Biology 46, 459–468.[CrossRef][Web of Science][Medline]
Ori N, Juarez MT, Jackson D, Yamaguchi J, Banowetz GM, Hake S. 1999. Leaf senescence is delayed in tobacco plants expressing the maize homeobox gene knotted1 under the control of a senescence-activated promoter. The Plant Cell 11, 1073–1080.
Pell EJ, Schlagnhaufer CD, Arteca RN. 1997. Ozone-induced oxidative stress: mechanisms of action and reaction. Physiologia Plantarum 100, 264–273.[CrossRef]
Pennell RI, Lamb C. 1997. Programmed cell death in plants. The Plant Cell 9, 1157–1168.[CrossRef][Web of Science][Medline]
Pennington MW, Thornberry NA. 1994. Synthesis of a fluorogenic interleukin-1 beta converting enzyme substrate based on resonance energy transfer. Journal of Peptide Research 7, 72–76.
Pontier D, Gan SS, Amasino RM, Roby D, Lam E. 1999. Markers for hypersensitive response and senescence show distinct patterns of expression. Plant Molecular Biology 39, 1243–1255.[CrossRef][Web of Science][Medline]
Quirino BF, Noh YS, Himelblau E, Amasino RM. 2000. Molecular aspects of leaf senescence. Trends in Plant Science 5, 278–282.[CrossRef][Web of Science][Medline]
Raff MC, Barres BA, Burne JF, Coles HS, Ishizaki Y, Jacobson MD. 1994. Programmed cell death and the control of cell survival. Philosophical Transactions of the Royal Society London B Biological Science 345, 265–268.[CrossRef]
Rano TA, Timkey T, Peterson EP, Rotonda J, Nicholson DW, Becker JW, Chapman KT, Thornberry NA. 1997. A combinatorial approach for determining protease specificities: application to interleukin-1 beta converting enzyme (ICE). Chemistry and Biology 4, 149–155.[CrossRef][Web of Science][Medline]
Sachs T. 1966. Senescence of inhibited shoots of peas and apical dominance. Annals of Botany 30, 447–456.
Schmid M, Simpson D, Gietl C. 1999. Programmed cell death in castor bean endosperm is associated with the accumulation and release of a cysteine endopeptidase from ricinosomes. Proceedings of the National Academy of Sciences, USA 96, 14159–14164.
Schmid M, Simpson DJ, Sarioglu H, Lottspeich F, Gietl C. 2001. The ricinosomes of senescing plant tissue bud from the endoplasmic reticulum. Proceedings of the National Academy of Sciences, USA 98, 5353–5358.
Smirnoff N. 1993. Tansley Review 52. The role of active oxygen in the response of plants to water-deficit and desiccation. New Phytologist 125, 27–58.[CrossRef][Web of Science]
Snow R. 1931. Experiments on growth and inhibition. II. New phenomena of inhibition. Proceedings of the Royal Soceity London, Series B 108, 305–316.
Snow R. 1932. Experiments on growth and inhibition. II. Inhibition and growth promotion. Proceedings of the Royal Soceity London, Series B 111, 76–105.
Solomon M, Belenghi B, Delledonne M, Levine A. 1999. The involvement of cysteine proteases and protease inhibitor genes in programmed cell death in plants. The Plant Cell 11, 431–444.
Steller H. 1995. Mechanisms and genes of cellular suicide. Science 267, 1445–1449.
Sun YL, Zhao Y, Hong X, Zhai ZH. 1999. Cytochrome c release and caspase activation during menadione- induced apoptosis in plants. FEBS Letters 462, 317–321.[CrossRef][Web of Science][Medline]
Thornberry NA, Lazebnik Y. 1998. Caspases: enemies within. Science 281, 1312–1316.
Tian R-H, Zhang G-Y, Yan C-H, Dai Y-R. 2000. Involvement of poly(ADP-ribose) polymerase and activation of caspase-3-like protease in heat shock-induced apoptosis in tobacco suspension cells. FEBS Letters 474, 11–15.[CrossRef][Web of Science][Medline]
Tiwari BS, Belenghi B, Levine A. 2002. Oxidative stress increased respiration and generation of reactive oxygen species, resulting in ATP depletion, opening of mitochondrial permeability transition and programmed cell death. Plant Physiology 128, 1271–1281.
Uren AG, O’Rourke K, Aravind L, Pisabarro MT, Seshagiri S, Koonin EV, Dixit VM. 2000. Identification of paracaspases and metacaspases: Two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma. Molecular Cell 6, 961–967.[Web of Science][Medline]
Valpuesta V, Lange NE, Guerrero C, Reid MS. 1995. Up-regulation of a cysteine protease accompanies the ethylene-insensitive senescence of daylily (Hemerocallis) flowers. Plant Molecular Biology 28, 575–582.[CrossRef][Web of Science][Medline]
Villa P, Kaufmann SH, Earnshaw WC. 1997. Caspases and caspase inhibitors. Trends in Biochemical Science 22, 388–393.
Wang X, Redziniak G, Bregegere F, Milner Y. 1998. Aged keratinocytes display increased levels of apoptosis- associated proteins, and increased susceptibility to apoptosis induction. Journal of Investigative Dermatology 110, 555–555.
Wilson CL, Shirras AD, Isaac RE. 2002. Extracellular peptidases of imaginal discs of Drosophila melanogaster. Peptides 23, 2007–2014.[CrossRef][Web of Science][Medline]
Woltering EJ, van der Bent A, Hoeberichts FA. 2002. Do plant caspases exist? Plant Physiology 130, 1764–1769.
Xu Y, Hanson MR. 2000. Programmed cell death during pollination-induced petal senescence in petunia. Plant Physiology 122, 1323–1333.
Xue D, Horvitz HR. 1995. Inhibition of the Caenorhabditis elegans cell-death protease CED-3 by a CED-3 cleavage site in baculovirus p35 protein. Nature 377, 248–251.[CrossRef][Medline]
Ye ZH, Varner JE. 1996. Induction of cysteine and serine proteases during xylogenesis in Zinnia elegans. Plant Molecular Biology 30, 1233–1246.[CrossRef][Web of Science][Medline]
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