Journal of Experimental Botany, Vol. 51, No. 345, pp. 721-730,
April 2000
© 2000 Oxford University Press
A Ca2+-dependent cysteine protease is associated with anoxia-induced root tip death in maize
1 Department of Crop Sciences, University of Illinois, Urbana, IL 61801–4730, USA
2 USDA/ARS, Soybean/Maize Germplasm, Pathology and Genetics Research Unit, Urbana, Illinois 61801, USA
Received 14 October 1999; Accepted 22 December 1999
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Abstract |
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Imposition of anoxia on maize (Zea mays cv. B73) seedlings for 48 h or longer led to the death of the root tip. The necrosis extended into the root axis during post-anoxic treatment, leading to the mortality of 30–50% of the seedlings. Using zymography, protease profiles in the root tissues of anoxic seedlings were studied. O2 deprivation for 24 h or longer repressed pre-existing protease activities and induced a novel soluble enzyme in the roots. The anoxia-induced protease (AIP) activity was predominant in the root apex at 24 h of anoxia and, subsequently, became the most abundant soluble activity in the root axis as well. The induction of AIP and its in vitro renaturation were Ca2+-dependent. Inhibitor sensitivity studies indicated that AIP is a cysteine protease. In SDS-acrylamide gels, the enzyme activity migrated as a 23.5 kDa polypeptide. The anoxic induction of the activity was repressed by cycloheximide treatment, suggesting that new protein synthesis was required for the AIP appearance. Excision of the root tip (de-tipping) before anoxia led to a superior recovery of seedlings from stress injury. De-tipped seedlings showed lesser root damage and an increased production of lateral roots compared to intact seedlings. Furthermore, the superior anoxia tolerance of de-tipped seedlings was associated with a decreased AIP activity. Thus, the appearance of AIP activity in the root tip at 24 h of anoxia was spatially and temporally associated with the root tissue death. These studies further indicate that the root tip elimination early during anoxia may provide an adaptive advantage.
Key words: Anoxia, root tip, cell death, cysteine protease.
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Introduction |
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Oxygen deprivation limits the growth and productivity of submerged plants. The magnitude of tolerance to anaerobiosis not only varies among different plant species (Crawford, 1992
; Kennedy et al., 1992) or cultivars within a species (Lemke-Keyes and Sachs, 1989), but also within different tissues of an individual plant (Johnson et al., 1989; Sachs, 1994; Ellis et al., 1999). Maize root tips are very sensitive to anoxia and die within a few hours (Roberts et al., 1984; Johnson et al., 1989). Root tips (or apices) are composed of tightly packed tissues with few, if any, intercellular spaces and hence suffer from restricted gaseous diffusion. In addition, the young, dividing cells in this region are metabolically-intensive with high O2 demand and, therefore, experience hypoxia even under normal oxygen partial pressures (Armstrong et al., 1991; Crawford, 1992; Andrews et al., 1993). Consequently, in submerged seedlings, root tip death may be a natural consequence of oxygen starvation and the attendant repression of substrate transport. A lot of attention has been given to strategies/mechanisms that prolong the anoxia tolerance of the primary root tip in young maize seedlings, as the tip of the primary root is considered to be very important for seedling establishment (for a review, see Drew et al., 1994). On the other hand, the hypothesis proposed here is that the death of the primary root tip under severe oxygen deficit can be a tolerance mechanism that eliminates dispensable but metabolically intense tissues, and redirects the limiting resources to prolong the survival of the root axis and the shoot. These two latter organs are essential for the post-anoxic re-growth and autotrophic life of the seedling. Hence, the goal in the present work is to test the proposal that the death of the primary root tip has an adaptive advantage, and to probe further the mechanisms that regulate the process. In this report, the time-course of root tip death initiation in the maize inbred, B73, was studied. To identify potential regulators of the cell death process, the changes in protease activities in the root apex and axis were followed. Increasing evidence indicates that proteases play an important regulatory role in the initiation of cell death (Chen and Foolad, 1997, and references therein), although other hydrolases (such as nucleases, lipases and cell wall-acting enzymes, e.g. cellulases, XET) may assume important roles in the later stages of cell lysis. Cysteine and serine proteases have been implicated in the cell death/injury induced by abiotic, biotic or developmental signals in plants (Schaffer and Fischer, 1988; Williams et al., 1994; Jiang et al., 1995; Stroeher et al., 1997; Del Pozo and Lam, 1998; Solomon et al., 1999; for reviews, see Beers, 1997; Hadfield and Bennett, 1997). The results of this study indicate that the root tip self-amputation has an adaptive role in the seedling anoxia tolerance and a Ca2+-dependent soluble cysteine protease may be involved in the death of the root tip. |
Materials and methods |
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Materials
Caryopses of inbred maize (Zea mays L.) line B73 were germinated and grown as described previously (Subbaiah et al., 1994b
). The root system of seedlings used in the study consisted of only a primary root (5–8 cm) and two small seminal roots (<10 mm). Only the primary root was utilized for this analysis and hence the terms ‘tip’ and ‘axis’ used throughout the text apply to the apical and distal portions of the primary root. Protease inhibitors were purchased from Calbiochem (La Jolla, CA) or Roche Molecular Biology (Indianapolis, IN). All other chemicals were from Bio-Rad (Hercules, CA) or Sigma (St Louis, MO) and were analytical grade.
Treatment of seedlings and preparation of root extracts
Anoxia was imposed by submerging the seedlings in gas-tight containers completely filled with flooding buffer (5 mM TRIS-Cl, pH 7.5, 250 mg amoxicillin+125 mg ampicillin l-1) as described earlier (Subbaiah et al., 1994b). In initial experiments, the submerged seedlings were incubated in an anaerobic system to create rapid and complete anoxia (Subbaiah et al., 1994a). The results did not vary between these two methods of imposing oxygen deprivation and, hence, the term ‘anoxia’ was used throughout this report. At selected time points of anoxic treatment, only the primary roots were harvested and used for enzyme extraction or frozen in liquid N2 for later use. Root tissue was ground in a buffer containing 62 mM TRIS-Cl pH 6.8, 10% glycerol and 2% Triton X-100 for total proteases and the extract was centrifuged at 10 000 g for 10 min. The supernatant was used for protease analysis. Soluble and membrane fractions were prepared from fresh, unfrozen tissue. Triton X-100 was omitted from the extraction buffer. The extract was centrifuged at 10 000 g for 10 min to pellet organelles and cell debris. The clarified extract was further centrifuged at 35 000 rpm for 90 min in a Ti45 rotor (Beckman-Coulter, Fullerton, CA). The supernatant represented the soluble fraction and the pellet was regarded as the membrane fraction. To test the effect of extracellular Ca2+on protease induction, maize seedlings were submerged in the presence of 2 mM of EGTA or CaCl2 and used for protease extraction. The effect of cycloheximide (CHI, a translational inhibitor) on protease induction was tested by adding the drug at 5 or 20 µg ml-1 in the submergence buffer. Control seedlings were incubated with methanol (used as solvent for CHI, up to 0.05% at final dilution). In experiments where protease profiles were analysed separately in the tip and axis, the root apex (‘tip’; up to 10–15 mm from the extreme end of the root including the root cap) was excised from the rest of the root (‘axis’), and processed separately.
Zymographic analysis of protease activities:
Protein concentration was estimated using DC (detergent-compatible)-Protein Assay kit from Bio-Rad. Protease activities were analysed by native or denaturing PAGE in gelatin-copolymerized acrylamide gels (modified from Fisher et al., 1989). Briefly, proteins (equal amounts in each lane, for all comparisons) were electrophoresed in 6–12% acrylamide gradient gels containing 0.033% gelatin. The gels were washed in 2.5% Triton X-100 and incubated overnight in assay buffer containing 50 mM TRIS-Cl, pH 7.5, 150 mM KCl, 10 mM CaCl2, 100 µM ZnCl2, and 0.05% Brij-50. The effect of Ca2+ was tested by omitting CaCl2 and adding 2 mM EGTA to the buffer. Sensitivity to protease inhibitors was tested by incubating the extracts at indicated concentrations of the inhibitors for 20 min at room temperature, before electrophoresis. Inhibitors were also added in the wash and activity buffers. Water-insoluble inhibitors (calpain inhibitor I, chymostatin and pepstatin) were dissolved in DMSO and used at concentrations indicated in the Results. As a control for these inhibitors, an appropriate concentration of the solvent (up to 5%) was tested on protease activity. Native-SDS two-dimensional gels were run as described previously (Lal et al., 1998).
In vitroanalysis of protease activities:
Protease activity was assayed spectrophotometrically using Azocoll as the substrate (Chavira et al., 1984).
Flood tolerance tests
Maize seedlings were submerged and post-anoxic survival as well as growth were measured as described earlier (Subbaiah et al., 1994b). To de-tip seedlings, the apical 15 mm region was excised under flooding buffer and discarded. The de-tipped seedlings were then submerged and tolerance tested.
Digitization and quantification of zymography results
Gels were photographed using Polaroid Instant photography (Type 665 or 667). The positives were scanned using a flat-bed scanner and stored as digitized images. The images were analysed using an NIH Image program (developed at the United States National Institute of Health and available on the internet at http://rsb.info.nih.gov/nih-image). The band intensities were measured, subtracted from background, integrated with areas and averaged.
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Results |
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Imposition of anoxia on maize seedlings for 48 h or beyond leads to the death of the root tip and the proximal axis
Three-day-old dark-grown maize (cv. B73) seedlings were submerged for different periods and were planted in the sand bench to recover. Seedlings were gently uprooted after 2 d and observed for root and shoot injury/re-growth. The seedlings when submerged had only primary roots and rudimentary seminal roots. Anoxia (i.e. complete submergence in sealed containers) rapidly suppressed all growth and developmental processes in maize seedlings. No new meristems were produced until after reoxygenation. Therefore, the terms root ‘tip’ and ‘axis’ refer to those of the primary root, throughout this report. Post-anoxic recovery of root and shoot growth was inversely proportional to the duration of anoxia, but the stress injury was particularly severe at 48 h of O2 deprivation, when roots showed necrosis of the tip and the elongation zone after reoxygenation treatment (Fig. 1
). The seedling recovery in this genotype was close to 100% until 24 h of anoxia and thereafter it declined (Table 1; Lemke-Keyes and Sachs, 1989). Furthermore, the tip growth resumed after re-aeration in seedlings submerged for 24 h or less (Fig. 1), although cell death indicators such as callose deposits and associated biochemical changes appeared in the tip within 24 h or shorter duration of anoxia (CC Subbaiah and MM Sachs, unpublished data). This indicated that the death process, although initiated before 24 h, became irreversible at 48 h of inundation. Even in 48 h submerged seedlings, tissue browning did not occur as long as seedlings were submerged, but was seen only after an exposure to air for at least 12 h, indicating that oxygen is required for the appearance of visual symptoms of the death process. In this report, the term root tip ‘death’ signifies the post-anoxic appearance of necrotic symptoms and growth cessation, while ‘survival’ is the lack of such symptoms and resumption of growth.
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Anoxia-induced changes in root protease activities of maize seedlings
Total collagenase (azocoll-hydrolysing) activities in root extracts from aerobic and submerged seedlings are shown in Fig. 2. Protease activity declined in the root under anoxia, particularly after 12 h and corresponded with the anoxia-induced growth-suppression. This was further supported by the recovery of protease activity within 24 h of re-oxygenation (see below). In seedlings submerged for 48 h, the activity was only 50% of the aerobic levels. Azocoll activity declined by only 18–20% in seedlings that remained under aerobic incubation for 48 h (Fig. 2).
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The activity profile as resolved by native-PAGE shows that a prominent gelatinase activity seen in the root axis of aerobic seedlings (marked by an arrowhead) rapidly declined during anoxia (Fig. 3A
). O2 deprivation increased a high molecular weight band in the root axis (indicated by the asterisk) and induced a faster migrating gelatinase species in the tip as well as the axis (indicated by an arrow). By 48 h of anoxia, the high molecular activity of the axis (marked by the asterisk) declined to a barely detectable level, while the fastest migrating species became the most predominant activity both in the tip and the axis. No activity in the range was detectable in aerobic extracts, even after 48 h of aerobic incubation (Fig. 3B), indicating that this may be a novel protease induced by anoxia. This activity, hereafter referred to as AIP (anoxia-induced protease), is the object of the present investigation.
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In root extracts of seedlings left under aerobic incubation for 48 h, a slow-migrating activity became intense (arrowhead, Fig. 3B
) indicating that total gelatinase activity increased substantially during aerobic growth. Apparently this species cannot utilize azocoll as the substrate, as total azocoll activity showed a small but consistent decline by 48 h from ‘zero’-time aerobic control (Fig. 2).
AIP is a soluble protease and migrates as a 23.5 kDa polypeptide in denaturing gels
When the extract was separated into soluble and microsomal fractions, AIP was recoverable from the soluble part of the extract with no detectable activity in the membrane faction (Fig. 4). In this report, it has not been determined if the enzyme is cytoplasmic or localized to any organelle. Zymography of anoxic and aerobic extracts in SDS-denaturing gels showed a greater diversity of activities than was found in native gels. This indicated that many protease species existing inactive in the root extracts were activated during renaturation in vitro (in addition to the superior resolving power of SDS-PAGE). There were activities that corresponded with the bands in native gels. However, in initial attempts, no activity in SDS-(or native/SDS 2-dimensional) gels could be matched with the AIP band comparable in its position or time-course in native gels. AIP activity was irreversibly denatured in SDS-containing gels run at or above the ambient temperature (above 20 °C, data not shown). Electrophoresis in cold (~10 °C) facilitated the renaturation of the enzyme (Fig. 5A). AIP migrated as a 23.5 kDa polypeptide, as was also confirmed by 2-dimensional native/SDS PAGE analysis (Fig. 5B). AIP activity was relatively greater in the tip region during early anoxia, but increased later in the axis as well (Fig. 5A).
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During the progression of anoxia, novel high molecular mass activities, i.e. >100 kDa appeared with different kinetics (Fig. 5A
). In the fractionation analysis, most of these activities were found to be associated with the microsomal fraction (data not shown). In the present study, the focus has been on AIP, the major soluble activity induced by anoxia.
AIP induction may require new protein synthesis
It was tested whether AIP is a novel protease whose synthesis is triggered by anoxia or, alternatively, whether it is a pre-existing enzyme that is activated by O2 deprivation in the root tissue. Cycloheximide (CHI), a translational inhibitor to repress de novo protein synthesis under anoxia, was used. In seedlings submerged in the presence of CHI (at 20 µg ml-1), AIP was totally repressed (Fig. 6A) and at a lower concentration of the inhibitor (5 µg ml-1), the activity was decreased by 75% Fig. 6B). These results indicated that the AIP synthesis was induced by O2 deprivation. The effect was fairly specific to AIP as all the major protease activities present in untreated seedlings, including a few novel ones, were detectable in extracts from CHI incubated seedlings.
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In vitro renaturation of AIP and its in vivo induction are Ca2+ dependent
It was investigated whether Ca2+ is really required for AIP activity, as the cation was routinely added to the assay buffer. In native gels, removal of Ca2+ by EGTA addition decreased the enzyme activity by ~90% (data not shown). In denaturing gels, AIP activity was totally abolished by EGTA and Ca2+ was necessary to activate the enzyme (Fig. 7A). Other cations tested, for example, Mg2+, Mn2+ or Zn2+ did not relieve the inhibition of EGTA on AIP activity (data not shown). Furthermore, the effect of Ca2+-removal on the anoxic induction of AIP was tested by adding EGTA to the submergence buffer. The in vivo induction of the protease was inhibited in the absence of extracellular Ca2+ (Fig. 7B), indicating that Ca2+ may signal AIP gene expression. However, EGTA did not affect the induction of other major proteases visible in the extracts from Ca2+-treated (Fig. 7B) or untreated seedlings (Fig. 3, lane T48 and A48), indicating that the inhibition of AIP was not due to a non-specific inhibition of protein synthesis by EGTA.
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AIP is a cysteine protease
The active site specificity of AIP was probed by testing the effects of class-specific inhibitors. E-64 (1.64 µM), a potent cysteine protease inhibitor completely blocked AIP activity (Fig. 8). Chymostatin (100 µM), an inhibitor of most of the cysteine proteases and the Calpain Inhibitor I (6 µM), a peptide inhibitor of animal calpain-type neutral cysteine proteases, decreased the activity by 66% and 83%, respectively. Pepstatin (1 µM), an inhibitor of aspartic proteases did not cause a decrease in AIP activity. Pefablock (0.4 and 2 mM), used as an inhibitor of serine proteases, but also known to act against certain type of cysteine proteases (Alonso et al., 1996), showed a mild inhibitory effect on AIP activity. These results suggest that AIP is a cysteine protease, functionally similar to the calpain type of Ca2+-dependent thiol proteases. Calpains have been reported to occur in a wide variety of organisms such as vertebrate and invertebrate animals, fungi and algae (for reviews, see Mykles and Skinner, 1990; Sorimachi et al., 1996; Johnson and Guttman, 1997). Results of many studies strongly suggest that cell death under ischemia and hypoxia in vertebrates is a result of calpain activation (Johnson and Guttmann, 1997).
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De-tipping decreases AIP activity and alleviates anoxic stress injury
To test if the apical part of the root is the origin and major source of AIP, the root tip (10–15 mm of the apex) was removed before seedlings were submerged. The AIP activity in de-tipped roots was compared with the activity in the root axes of intact seedlings (Fig. 9A). De-tipping reduced the enzyme activity by ~50% in the root axis of seedlings submerged for 24 h and about 30% at 48 h of anoxia. This indicated that the tip or tip-associated processes may play a role in the induction of AIP even in the axis region. The dynamics of AIP activity were also tested soon after reoxygenation. Re-aeration decreased the AIP activity in the root axis (the tip, being necrotic, was discarded) by greater than 50% within 24 h (Fig. 9B).
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The aim here is also to determine if the root tip removal before submergence would lead to superior anoxic tolerance. The post-anoxic survival of de-tipped and intact maize seedlings was compared after they had been submerged for different periods. Removal of the root tip did not affect the survival or growth of aerobic seedlings except that there was an increased partitioning of biomass into the roots in comparison to intact seedlings (Table 1
), most likely due to a loss of root apical dominance. This trend continued until 12 h of anoxia. However, better root growth in aerobic and early anoxic seedlings did not lead to a superior shoot growth but actually reduced the shoot biomass. However, later on, de-tipping proved to be clearly advantageous to maize seedlings. As shown in Fig. 10, de-tipped seedlings showed greater stress recovery both in terms of percentage survival (Fig. 10A) and biomass accumulation in the root as well as in the shoot (Fig. 10B) after 2 weeks of re-aeration. De-tipped seedlings showed very little root necrosis and thereby produced lateral roots throughout the root axis. De-tipping also reduced shoot necrosis, leading to a faster emergence as well as expansion of primary leaves, within 48 h of re-oxygenation (Fig. 10C). In intact seedlings submerged for 48 h or longer, most of the root axis was severely necrotic and produced very few laterals in its proximal region (Fig. 10C). Furthermore, primary leaf emergence was also delayed in intact seedlings (Fig. 10C). It is hypothesized that the removal of the primary root tip before imposing anoxia has allowed maize seedlings to redirect the limiting resources (such as ATP, pyridine nucleotides and respiratory substrates: Roberts et al., 1984; Saint-Ges et al., 1991; Ricard et al., 1994) for the protection of the primary root axis and shoot from anoxic injury. This has apparently led to a rapid resumption of growth after re-aeration in de-tipped seedlings.
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The primary root tip is a dispensable energy drain in anoxic maize seedlings
The seedling primary root plays a brief but important role in the life cycle of maize, as in the case of most herbaceous monocotyledons. In most maize genotypes, the seminal roots grow and contribute to nutrient and water absorption, within the first few weeks after germination. After a month of seedling emergence, the seminal root system (the primary and seminal roots) assumes less importance and frequently decays following the development of the nodal or permanent root system (Feldman, 1994
). The primary root tip is needed for even a shorter duration, i.e. only during the first few days of germ growth, to establish seedling orientation in space and to produce initially tissues involved in water/nutrient uptake. The root apex is metabolically very intense and thus has greater O2 demand than the rest of the primary root (Armstrong et al., 1991; Lin and Lin, 1992). In young maize seedlings subjected to prolonged anoxia, the primary root tip appears to serve no perceptible function essential for seedling survival. Instead, it may be a non-essential sink which, if eliminated, can conserve limiting resources. The conserved substrate and energy can usefully be redirected to prolong the maintenance of organs essential for growth after re-aeration. These studies show that excision of the primary root tip was not detrimental to either the aerobic or anoxic growth of young maize seedlings (Table 1). On the contrary, removal of the root tip before imposing anoxia alleviated the stress injury and prolonged the tolerance of maize seedlings to oxygen deprivation. Thus, the death of the metabolically intensive root tip, induced by prolonged anoxia, served as a tolerance mechanism against the severe energy starvation known to occur under O2 deprivation (Ricard et al., 1994). Recently, Zeng et al. (Zeng et al., 1999) compared the anoxic tolerance of excised root tips with that of intact seedlings, in three different maize genotypes. Maize variety Oh43 which has the least anoxic tolerance in excised root tips shows greater whole seedling tolerance to anoxia than NK508 and W22, indicating that the early death of the primary root tip confers superior flooding tolerance in young maize seedlings. The natural ‘de-tipping’ of the primary root may be a genetically controlled flooding tolerance mechanism in plants, although the importance of the root tip during normal growth and development is indisputable.
Is AIP an initiator or executor of the root tip death?
AIP is a Ca2+-dependent soluble cysteine protease that was inducible in the root apical tissue within 24 h of anoxia. Its temporal precedence to the root necrosis and spatial distribution within the root indicate that AIP may play an important role in the initiation of root tip death. Furthermore, a decrease in AIP activity as well as a greatly reduced root necrosis in de-tipped seedlings suggests that the enzyme may serve as a molecular switch that turns on the cell death process. However, its continued presence, albeit at reduced levels, after re-oxygenation does not rule out that it may serve as an executor of cell degradative processes. As root necrosis in anoxically-treated maize seedlings continued even after reoxygenation, the continued presence of AIP activity in re-aerated seedlings may not be totally surprising. AIP was the only detectable gelatinase, which showed sensitivity to cysteine protease inhibitors (including calpain inhibitor 1) and also Ca2+ dependence, suggesting that the enzyme may be functionally analogous to animal calpains. Calpains are regulated by Ca2+ as well as cellular redox state (by virtue of having cysteine in the active site, they are inhibited by oxidants) and initiate hypoxic cell death in vertebrates (Johnson and Guttmann, 1997). It is necessary to identify the molecular targets of AIP and events downstream to its induction in order to understand whether the enzyme has a role in cell death. Furthermore, other proteases (particularly those, which cannot degrade gelatin) cannot be ruled out for their role in the root tip death.
With the present data, it is not possible to compare AIP with other proteases that have previously been identified from maize or other species. AIP is similar in its size and active site to the cysteine protease implicated in the programmed cell death during tracheary element differentiation in Zinnia elegans (Ye and Varner, 1996). However, it is not known if the zinnia enzyme requires Ca2+ for its activity. At the same time, AIP is different from ACDP, a Ca2+-dependent cysteine protease partially purified from Arabidopsis root tissue, in its size and sensitivity to calpain inhibitors (Safadi et al., 1997).
Cytosolic Ca2+ changes may be involved in the initiation of cell death in anoxic root tips
Earlier work showed that an elevation of cytosolic Ca2+ triggers gene activation and tolerance to O2 deprivation (Subbaiah et al., 1994a, b). Ca2+-release from intracellular stores is critical for anoxic tolerance during the first few hours of anoxia (Subbaiah et al., 1994b) and seedling survival is compromised in the absence of extracellular Ca2+, beyond 12 h of anoxia (CC Subbaiah, unpublished data). Subsequently, it has been shown that hypoxia-induced cell death during aerenchyma formation is also a Ca2+-dependent process (He et al., 1996). Thus, cytosolic Ca2+-rise appears to switch on the co-ordinate gene expression induced under oxygen deprivation in maize roots. The current work shows that the AIP induction is inhibited by EGTA when added to the submergence buffer (Fig. 7C) and thus its synthesis and cell death initiation may depend on elevated cytosolic Ca2+ levels in the root tip cells. Furthermore, the inhibition of AIP induction by the translational inhibitor, cycloheximide, gives a preliminary indication that this protease may be one of the gene products whose synthesis is co-ordinately activated under anoxia (Sachs et al., 1980).
In summary, these studies show that the death of the primary root tip in young maize seedlings between 24–48 h of anoxia may be regulated by AIP, a Ca2+-dependent cysteine protease. Further, this tissue-suicide, if initiated earlier, appears to provide greater anoxia tolerance to submerged seedlings.
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Acknowledgments |
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We thank Dr Donald Ort, University of Illinois, Urbana-Champaign and Dr Douglas Bush, University of California-Santa Barbara for critically reading the manuscript. Sincere thanks are also due to Professor Theodore Hymowitz for his excellent co-operation and facilities during this work. This work is funded by NRICGP- grant No. 96–35100–3143 from the USDA to MMS and CCS.
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Notes |
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3 To whom correspondence should be addressed. Fax: +1 217 333–6064. E-mail: subbaiah{at}uiuc.edu
4 Present address: Pioneer Hi-Bred International Inc., PO Box 1004, Johnston, IA 50131, USA.
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