Annals of Botany 91: 195-204, 2003
© 2003 Annals of Botany Company
Assessment of Enzyme Induction and Aerenchyma Formation as Mechanisms for Flooding Tolerance in Trifolium subterraneum ‘Park’
1 Département de Biologie, Faculté des Sciences de Tunis, 1060 Tunis, Tunisia and 2 UMR de Physiologie et Biotechnologie Végétales, BP 83, 33883 Villenave d’Ornon Cedex, France
* For correspondence. Fax + 33 (0) 557 122541, e-mail ricard{at}bordeaux.inra.fr
Received: 29 June 2001; Returned for revision: 23 August 2001; Accepted 16 October 2001
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSLITERATURE CITED |
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The objective of this study was to evaluate the role of enzyme induction and aerenchyma formation in prolonged tolerance to soil flooding in a variety of underground clover (Trifolium subterraneum ‘Park’) previously selected for resistance. Seedlings were grown in hydroponic tanks, initially with aeration for 3 weeks and subsequently in the absence of aeration for up to 3 weeks. After 1 h in the absence of aeration, the oxygen concentration in the hydroponic medium had decreased to 1·5 %. During the 3 weeks of extreme oxygen deficiency, primary roots died and were replaced by considerable numbers of adventitious roots. Activities of many glycolytic and fermentative enzymes increased in adventitious roots. Excised adventitious roots were capable of immediate induction of ethanol in the absence of lactate production, in association with energy charge higher than that in excised roots of aerobically maintained controls. Energy charge was even higher when measured in adventitious roots in planta. Interestingly, haemoglobin protein could be correlated with energy charge. Aerenchyma was readily visualized in adventitious roots by optical microscopy of longitudinal and transverse sections. We conclude that avoidance of root anoxia via aerenchyma is the major mechanism for prolonged root tolerance in Trifolium subterraneum ‘Park’.
Key words: Trifolium subterraneum, clover, flooding, hypoxia, anoxia, acclimation, energy metabolism, aerenchyma, roots, haemoglobin, endopeptidases.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSLITERATURE CITED |
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Trifolium species are small-seeded pasture legumes with varying tolerance to waterlogging. For instance, Trifolium tomentosum (L.) has been reported to be more tolerant to waterlogging than T. glomeratum (L.) (Gibberd and Cocks, 1997). Thus, although both species form lateral roots in a typical response to low oxygen conditions (Rogers and West, 1993; Gibberd and Cocks, 1997), primary root extension, as well as the relative growth rate of the whole root system, was inhibited to a greater extent in T. glomeratum (Gibberd et al., 1999). An earlier study of T. fragiferum and T. repens showed that roots produced during waterlogging had a higher porosity and this was associated with the lysigenous breakdown of cortical cells (Rogers and West, 1993). Tolerance of oxygen-deficient waterlogged soils in a number of plant species has been shown to be associated with the development of aerenchyma and internal oxygen movement from shoot to roots (Laan et al., 1990; Thomson et al., 1990, 1992; Colmer et al., 1998). A recent study sought to evaluate the role of aerenchyma in the differing tolerance to waterlogging of T. tomentosum compared with T. glomeratum (Gibberd et al., 1999). Constitutive porosity in aerated T. tomentosum roots was greater than in T. glomeratum roots. Although both species responded to root-zone hypoxia by increasing root porosity to similar extents (1·6-fold), higher constitutive porosity in T. tomentosum resulted in a two-fold higher root porosity after 21 d of hypoxia. The root porosity was further shown to provide a low resistance internal pathway for oxygen diffusion in both species. Increased root porosity leading to enhanced internal oxygen supply is suggested to be part of the basis of increased tolerance to waterlogging (Gibberd et al., 1999).
The susceptibility of a variety of underground clover (T. subterraneum ‘Clare’) to waterlogging has been reported previously (Francis and Devitt, 1969; Francis and Poole, 1973) and was confirmed in a study of the effect of concurrent root zone salinity and hypoxia in several Trifolium species (Rogers and West, 1993). Rogers and West (1993) correlated the presence of aerenchyma in the adventitious roots which emerge from the stem at the water surface with the superior tolerance of T. michelianum, T. isthmocarpum and T. fragiferum compared with T. subterraneum and T. purpureum. Unlike T. tomentosum and T. glomeratum, T. subterraneum differs in its ability to form adventitious roots (Aschi-Smiti et al., 2002). Of the four different Trifolium subterraneum L. varieties (Larisa, Clare, Montbaker, Park) tested, Park was judged to be the most resistant to waterlogging under glasshouse conditions, in accord with the development of larger numbers of adventitious roots. Resistance was evaluated by immersion for 10 d at the second trifoliate leaf stage. Under such conditions, Park was the only variety seemingly improved by immersion, judging from its better shoot aspect and increased shoot yield compared with normally watered seedlings. The observed concomitant disappearance of primary roots and the production of new adventitious roots was suggested to be part of the basis of tolerance to waterlogging (Aschi-Smiti et al., 2002). In this paper we investigate whether and how energy metabolism is improved in roots produced under low oxygen conditions and whether anaerobic protein (ANP) and/or haemoglobin induction could play a role in improved energy metabolism. We also show the presence of aerenchyma in roots produced under low oxygen conditions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSLITERATURE CITED |
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Plant material and growth conditions
Seeds of Trifolium subteranum ‘Park’ [kindly provided by the Institut National de la Recherche Agronomique de Tunisie (INRAT)] were disinfected for 15 min in a commercial solution of sodium hypochlorite (1 : 3 dilution), rinsed thoroughly and soaked overnight in distilled water before germination in the dark at room temperature between filter paper imbibed with S/4 (mineral solution S diluted four-fold; Saglio and Pradet, 1980). Five days after germination, 40–50 seedlings were transferred for hydroponic culture to 20 l plastic tanks filled with S or Algospeed solution containing N : P : K : Mg (13 : 13 : 24 : 3 g l–1) and continuously sparged with air. Plants were supported on floats with shoots in the air and roots plunging into the mineral solution. Plants were maintained in a growth chamber under controlled conditions (15 h at 25 °C/9 h at 20 °C light/dark regime; irradiance of 150–250 µmol m–2 s–1; 80 % humidity) for 3 weeks. The mineral solution (pH 5·8) was renewed every week, by which time the pH of the solution had decreased to 5·3–5·4.
Treatments
Five-day-old seedlings.
Seedlings were completely immersed in S/4 mineral solution which was either bubbled for 36 h with air or with 3 % oxygen in nitrogen. At harvest, seedlings were separated into root tips, root axis and hypocotyls.
Three-week-old plants.
Aeration of hydroponic tanks was arrested for 15 or 21 d in the hypoxic treatment or continued for the same length of time in aerobic controls. Measurements with a Clarke electrode showed 21 % oxygen in aerated solutions and 1·5 % oxygen in non-aerated solutions after 1 h. Plants were transferred weekly to fresh nutrient solution S (pH 5·8) previously bubbled with nitrogen for 30 min. At harvest, roots were washed in distilled water. As previously reported (Aschi-Smiti et al., 2002), it was observed that plants grown in such oxygen-deficient conditions developed many new adventitious roots, while the primary root system died and became detached from the plant. The root system of aerated plants consisted of primary and lateral roots.
Survival and energy charge determination
Survival of anoxia was evaluated as the capacity to increase ATP levels when tissues submitted to extreme oxygen deficiency were returned to air. Between 100 and 200 root tips from 5-d-old seedlings or 2 g f. wt of root segments from 30-d-old plants were placed in 50-ml syringes containing 10 ml S/4 and 250 µg ml–1 cefotaxime supplemented with 100 mM sucrose. The syringes with their rubber puncture caps were fitted with needles on rubber vacuum tubes flushed with nitrogen. After various incubation times in anoxia, triplicate samples consisting of ten root tips or about 50 mg f. wt of root segments were transferred to air to allow respiration to resume and ATP levels to increase in living cells. ATP was assayed by a bioluminescent luciferin–luciferase reaction as described by Saglio et al. (1988). This method has proven to be reliably correlated to viability (Xia and Saglio, 1992).
For adenylate energy charge (AEC) determinations in excised tissue, root tips of ten 5-d-old seedlings or about 50 mg f. wt of root segments from 30-d-old plants were placed in tubes flushed with a humidified stream of nitrogen. After 1 h of anoxic treatment the tubes were placed in liquid nitrogen to quick freeze the roots in the absence of contact with air. The frozen tubes were stored at –20 °C and nucleotides were extracted for estimation of ATP, ADP and AMP by bioluminescence. For AEC determinations in attached roots, plants were placed in containers on a floater with aerial parts in air and roots immersed in nutrient medium S flushed with oxygen-free N2. After 1 h of anoxic treatment the entire root system was transferred to liquid nitrogen. Roots were cut into segments while still frozen and kept at –20 °C until extraction of nucleotides for AEC determinations as described above. AEC was calculated as follows: AEC = (ATP + 0·5 ADP)/(ATP + ADP + AMP) (Pradet and Raymond, 1983).
Ethanol production
Root segments (500 mg f. wt) from 30-d-old plants were placed under anoxia in sealed syringes containing 10 ml of S/4 and 250 µg ml–1 cefotaxime supplemented with 100 mM sucrose as described above. Triplicate samples were removed at the indicated times of imposed anoxia and the concentration of ethanol was determined enzymatically as previously described (Saglio et al., 1980).
Glycolytic and fermentative enzyme activities
Root tips of 5-d-old seedlings, segments of adventitious roots, or segments of lateral and primary roots were homogenized in twice their fresh weight of 50 mM Tris (pH 7·5), 10 mM Na borate, 5 mM dithiothreitol (DTT), 15 % (v/v) glycerol, 1 % (w/v) bovine serum albumin, 0·1 % (v/v) Triton X-100 and 3 % (w/v) polyvinyl polypyrrolidone (PVP). The brei was clarified by centrifugation for 5 min in an Eppendorf centrifuge and desalted on spin columns as described by Bouny and Saglio (1996). Near complete recovery of protein from such spin columns is routinely obtained. Soluble proteins were quantitated on samples homogenized in the same buffer without BSA. Protein contents of root extracts from hypoxically treated (HT) or normoxically treated (NT) seedlings or plants were comparable (1·8 ± 0·5 and 1·9 ± 0·8 mg protein g–1 f. wt for HT and NT seedlings and 4·2 ± 1·2 and 4·6 ± 1·2 mg protein g–1 f. wt for HT and NT plants).
Activities of hexokinase (HK), fructokinase (FK), lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH) were assayed spectrophotometrically at 25 °C and pH 7·5 as described by Bouny and Saglio (1996). Activities of neutral invertase (INV) and sucrose synthase (SuSy) were measured successively by spectrophotometry at pH 7, essentially according to the method of Nguyen-Quoc et al. (1990).
Measurement of proteolytic activity
Fresh tissue (250–500 mg) was homogenized in a mortar at 4 °C with four times the fresh weight of extraction medium [50 mM Tris–HCl, pH 7·5, 5 mM ß-mercaptoethanol and 0·5% (v/v) PVP]. The homogenate was centrifuged for 15 min at 27 000 g and the supernatant was assessed for endopeptidase and chymotrypsin-like activities. Endopeptidase activities (against azocasein) were determined as described previously (James et al., 1993). Chymotrypsin-like activities were measured using N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin (Succ-Leu-Leu-Val-Tyr-AMC) as a substrate, and essentially as described by Brouquisse et al. (1998). Protease inhibitors were prepared as the following stock solutions: E-64 (10 mM) and Na2-EDTA (0·5 M) in water, phenyl methyl sulfonyl fluoride (PMSF, 0·2 M) in ethanol, 1–10 phenantroline (0·2 M) and pepstatin (5 mM) in methanol, 3–4 DCI (dichloroisocoumarin, 10 mM) and chymostatin (10 mM) in dimethylformamide. Inhibitors were first preincubated for 15 min at ambient temperature with protein extracts prior to substrate addition, and activities were measured as described above. Control assays were carried out with the corresponding solvent.
Electrophoresis and immunoblots
Proteins separated on SDS–12·5 % polyacrylamide gels were transferred to nitrocellulose membranes. Blots were probed with a polyclonal antiserum that recognized barley haemoglobin protein (kindly donated by R. Hill). Immuno detection was carried out by coupling the haemoglobin antisera with anti-rabbit IgG–alkaline phosphatase conjugate and visualization using BCIP (5-bromo-4-chloro-3-indolylphosphate) and NBT (nitroblue tetrazolium).
Cytology
Using a binocular microscope, 1 mm samples were excised from the apical meristematic region of the primary root of 5-d-old seedlings subjected to 36 h of hypoxia or normoxia and from adventitious roots produced by 30-d-old plants during a 15 d hypoxic treatment. One millimetre samples were also obtained from the hypocotyl of 30-d-old plants following a 15 d period of hypoxic or normoxic growth. Immediately after excision samples were placed in 6 % glutaraldehyde in 0·2 M sodium cacodylate (pH 7·4) for 45 min at 4 °C, then in 1 % osmium tetroxide in 0·1 M veronal (pH 7·4) for 45 to 60 min, as described by Sabbatini et al. (1963). Samples were fixed in Spurr’s resin (Sigma) and sliced to a thickness of 0·5 µm. The semi-fine slices were placed in 1 % toluidine blue for 15 min, then washed with tap water followed by distilled water. The fixed and stained samples were examined with a light microscope (Leica).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSLITERATURE CITED |
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Activities of glycolytic and fermentative enzymes
Roots from a variety of plants, including cereals (maize, rice) and tomato are capable of increasing their resistance to anoxic shock during exposure to decreasing oxygen levels (cf. Saglio et al., 1999). During ‘hypoxic acclimation’, mechanisms are activated that allow a better regulation of cytosolic pH (Roberts et al., 1985; Xia et al., 1995) and the maintenance of glycolytic flux at a level sufficient to supply the ATP necessary for cell survival (Xia et al., 1995). The latter strategy is invariably accompanied by an increase in the activities of many of the glycolytic and fermentative enzymes, the so-called hypoxically induced proteins (Saglio et al., 1999). An anaerobic response of this kind is an indicator of metabolic adjustment allowing the maintenance of a higher glycolytic flux, compatible with survival. To determine whether a response of this nature plays a role in the flooding tolerance of T. subterraneum, we measured the activities of several glycolytic and fermentative enzymes in extracts from root tips of 5-d-old seedlings following 36 h of hypoxic or normoxic treatment. We also measured the same enzyme activities in extracts from adventitious roots produced by 30-d-old plants following 15 d of HT. The latter were compared with activities in extracts from the root system (consisting of lateral and primary roots) of 30-d-old plants after 15 d of additional growth in aerated medium (NT). Table 1 shows that the activities of the enzymes implicated in the first steps of sucrose utilization were either unmodified or even decreased during HT in root tips of 5-d-old seedlings. Of the fermentative enzymes studied ADH, but not LDH, increased in activity, probably reflecting the universal nature of low oxygen induction of the Adh gene. In contrast, a typical hypoxic response was observed in adventitious roots produced by 30-d-old plants. FK and SuSy activities increased as did the activities of both LDH and ADH.
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Effect of increased enzyme activities on lactate and ethanol fermentation in adventitious roots
In maize, rice and tomato roots, increased glycolytic and fermentative enzyme activities subsequent to HT were accompanied by an increased ability to produce ethanol, decreased lactate production and survival for longer periods under complete deprivation of oxygen (anoxia) (cf. Saglio et al., 1999). To study these parameters in T. subterraneum ‘Park’, adventitious roots produced during 15 d of hypoxic treatment were excised, cut into segments and transferred to anoxia in medium supplemented with sucrose. Figure 1 shows that ethanol was immediately produced although lactate production remained constantly low. As is the case for 2-d-old rice seedlings, activation of ethanol synthesis is not preceded by lactate synthesis (Rivoal et al., 1989). The rate of ethanol production remained significant for 6 h but then stopped almost completely. Root tissue was still alive up to 10 h, as shown in Fig. 1B, so the arrest of glycolysis must be due to factors other than cell death. In spite of ample sucrose in the incubation medium, it is likely that an inability to import sucrose from the external medium under conditions of low energy leads to carbon exhaustion. Such is the case in root segments from HT tomato (Germain et al., 1997). Sucrose, but not glucose, allows ethanol production to continue beyond the period in which internal sugars have been shown to be available (Germain et al., 1997).
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Adenylate energy charge indicates improved energy metabolism in adventitious roots
Under anoxic conditions, higher AEC levels are an indication of improved energy metabolism (Saglio et al., 1980; Pradet and Raymond, 1983) and have been correlated with improved tolerance and survival. Since 5-d-old seedlings are extremely intolerant to anoxia, even after hypoxic treatment, and are also unable to induce glycolytic and fermentative enzyme activity during hypoxia, it was no surprise that both NT and HT root tips had similar AEC levels (Table 2). However, adventitious root segments from plants grown for 15 d in hypoxic conditions had a higher AEC than root segments of aerobically grown plants, confirming metabolic acclimation as indicated by higher enzyme activities and enhanced ethanol production. AEC was even higher in attached adventitious roots, suggesting additional mechanisms for energy production in the latter.
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According to data obtained with maize root tips (Saglio et al., 1988; Xia and Saglio, 1992; Bouny and Saglio, 1996), a rise in AEC in anoxia from values close to 0·4 in NT root tips to 0·6 in HT root tips corresponds to a doubling of the rate of glycolysis. Accordingly, the increase in AEC of our excised clover root segments from 0·4 to 0·6 is thought to be roughly equivalent to a doubling of the rate of ATP production by NT plants. In NT plants under anoxia, where ATP is produced essentially by glycolysis, this is equivalent to 2 mol ATP per mol glucose consumed. In adventitious roots submitted to anoxia in planta, oxygen transported from shoots increases the AEC to 0·7. By deduction from measurements of ATP/ADP as a sole function of pO2 in maize primary roots (Saglio et al., 1983), it can be estimated that such an increase in AEC corresponds to the amount of ATP produced at pO2 close to 1 %, which allows a respiratory rate of about 30 % of the maximum, or 12 mol ATP per mol glucose consumed. The small amount of oxygen reaching root tissues significantly improves their energy status, allowing their survival in the absence of external oxygen. In addition, the two-fold increase in glycolytic rate following hypoxic acclimation also contributes to the improved energy status by permitting more efficient use of carbohydrates for energy production.
Haemoglobin and energy metabolism
Non-symbiotic haemoglobin protein is probably ubiquitous and has variously been suggested to be an oxygen sensor, a transporter of oxygen (Appleby et al., 1988; Bogusz et al., 1990) or even to play a role in energy metabolism as an NADH oxygenase (Hill, 1998). Immunoblots of proteins from root tips, root axes and hypocotyls of 5-d-old seedlings hypoxically or normoxically treated, and from roots produced by 30-d-old HT and NT plants were probed with antiserum to barley haemoglobin. Figure 2 shows that haemoglobin protein is easily detected in the hypocotyl and root axis but is barely seen in the root tip of 5-d-old HT seedlings and is not seen at all in NT seedlings. Low levels of haemoglobin protein are detected in roots produced after 15 or 21 d of NT by 30-d-old plants. Levels are much higher in roots of HT plants. The correlation between haemoglobin levels and tissues with improved AEC lends support to a role of haemoglobin proteins in energy metabolism.
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Effect of hypoxia on aerenchyma formation in primary and adventitious roots
The hypocotyl and root axis of HT 5-d-old seedlings are comparable with those of NT control seedlings with no evidence for cellular separations (results not shown). However, comparisons of longitudinal sections through the primary root meristem of NT (Fig. 3A and B) and HT (Fig. 3C and D) seedlings show several modifications associated with hypoxic treatment. In HT root meristems, the cytoplasm is seen to be contracted, fine filaments connect the cell wall with the cytoplasm, and cell files are perturbed (Fig. 3C and D). These images are reminiscent of ‘plasmolysis’ previously described by Morisset (1978) in tomato roots submitted to prolonged oxygen deprivation.
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Figures 4 and 5 show that 15 d of hypoxia resulted in perturbations in the hypocotyls as well as in the root axis and meristems of 30-d-old plants. The envelope of uniform appearance in well-aerated hypocotyls (Fig. 4A and B) is torn or entirely absent in hypoxic hypocotyls (Fig. 4C). The cortical zone has numerous lacunae and adventitious root buds are formed from the central cylinder (Fig. 4D). Longitudinal views of the adventitious root meristem and axis shown in Fig. 5 also show evidence of ‘plasmolysis’, as seen in the meristematic region of primary roots from HT 5-d-old seedlings. In addition, large gaps are seen in the cortical parenchyma (Fig. 5A and B), forming longitudinal spaces separating files of completely differentiated cells. These cells are filled entirely by the vacuole, with what is left of the cytoplasm being pushed against the cell wall (Fig. 5B). Within the cell spaces, deformed cells (Fig. 5B) and fragments (Fig. 5D) can be observed.
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As expected, aerenchyma extends through shoots to the very tips of the adventitious roots examined, enhancing transport of oxygen from shoot to root. Oxygen transport from shoot through adventitious roots has been visualized directly (Aschi-Smiti et al. 2002).
Changes in endoproteolytic activities associated with hypoxically pretreated roots
The selective death of cells to form the gas-filled spaces between surviving cells, generally thought to be the origin of aerenchyma, should logically be associated with increased and/or different proteolytic activities. In plant cells, protein breakdown is mediated by different proteolytic systems associated with vacuolar, nuclear, cytosolic and organellar compartments (Vierstra, 1993). Total endopeptidase activities, which are principally vacuolar, were measured using azocasein as substrate. They were similarly low in root tips of both NT and HT 5-d-old seedlings (Fig. 6). Activities rose dramatically in root segments from 30-d-old plants and were higher in the lateral and primary roots of NT plants compared with the adventitious roots of HT plants. Serine, cysteine, aspartic and metallo-proteases can be distinguished among the endopeptidases through the use of inhibitors. Table 3 shows that E-64, a cysteine protease inhibitor, inhibits HT but not NT activities, suggesting that about 30 % of the endopeptidase activity in HT root tips is due to cysteine proteases. The equivalent effect on NT activities with PMSF and 3–4 DCI suggests that about 50–60 % of the endopeptidase activity in NT root tips is due to serine proteases. The weak inhibition observed with 1–10 phenantroline and pepstatin indicates that metallo- and aspartic proteinases both represent about 10 % of the endopeptidase activities in NT and HT root tips. Root segments from 30-d-old plants were analysed in the same way. E-64 had no effect on endopeptidase activities, whereas PMSF and 1–10 phenantroline inhibited serine and metallo-protease activities, respectively, in NT and HT roots to similar residual levels. However, chymostatin, a serine/cysteine protease inhibitor, inhibited NT but not HT activities, suggesting the presence of a different serine protease in NT roots. Taken together, the results suggest changes in protease composition which are, in part, attributable to developmental changes but which are also associated with hypoxic treatment. In animal tissues, hypoxia has been shown to induce nuclear accumulation of proteasome (Ogiso et al., 1999). Although we were unable to detect changes in either chymotrypsin activity (associated in other plants with the proteasome; Brouquisse et al., 1998) or in 20S proteasome levels (results not shown), we cannot rule out proteasome involvement in hypoxic induction of lysigenous aerenchyma without further experiments.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSLITERATURE CITED |
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The primary roots of very young T. subterraneum ‘Park’ seedlings (5 d old) are incapable of metabolic acclimation and excised root tips die after only 15 h of anoxic incubation. However, adventitious roots produced during 15 d of hypoxia by 30-d-old plants show signs of metabolic acclimation: induction of enzymes implicated in sucrose metabolism (SuSy, FK) and fermentation (LDH, ADH), enhanced ethanol production, and improved energy charge in association with haemoglobin induction. Energy charge is even higher when measured in adventitious roots in planta and is attributed to ATP production by aerobic respiration. Aerenchyma extending from shoots to roots can be visualized by light microscopy in hypoxically induced adventitious roots but not in lateral and primary roots of control plants. The development of aerenchyma and their expected involvement in oxygen transport is probably the major mechanism for long-term waterlogging tolerance of the Park variety of T. subterraneum.
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ACKNOWLEDGEMENTS |
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We thank Dr E. Bizid (Université de Tunis, Tunisia) for her dedicated support of this work and T. Menu (University of Bordeaux II, France) for his efficient technical assistance.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSLITERATURE CITED |
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E. J. W. VISSER, L. A. C. J. VOESENEK, B. B. VARTAPETIAN, and M. B. JACKSON Flooding and Plant Growth Ann. Bot., January 2, 2003; 91(2): 107 - 109. [Abstract] [Full Text] [PDF]
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