Journal of Experimental Botany, Vol. 54, No. 391, pp. 2363-2373,
October 1, 2003
© 2003 Oxford University Press
Anoxia tolerance in rice seedlings: exogenous glucose improves growth of an anoxia-‘intolerant’, but not of a ‘tolerant’ genotype
Received 21 February 2003; Accepted 25 June 2003
School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley 6009 WA, Australia
* To whom correspondence should be addressed. Fax: +61 8 9380 1108. E-mail: tdcolmer{at}cyllene.uwa.edu.au
Definition: Sugar accumulation ratio (embryo/endosperm): sugar concentration in embryo divided by sugar concentration in endosperm.
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Abstract |
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This study demonstrated that, in rice seedlings, genotypic difference in tolerance to anoxia only occurred when anoxia was imposed at imbibition, but not at 3 d after imbibition. When seeds were imbibed and grown in anoxia, IR22 (anoxia-‘intolerant’) grew much slower and had lower soluble sugar concentrations in coleoptiles and seeds than Amaroo (anoxia-‘tolerant’), while Calrose was intermediate. After 3 d in anoxia, the sugar concentrations in embryos and endosperms of anoxic seedlings were nearly 4-fold lower in IR22 than in Amaroo. Sugar deficit in the embryo of IR22 is presumably due to the limitation of sugar mobilization rather than the capacity of transport as shown by similar sugar accumulation ratios of 1.8 between embryo and endosperm in IR22 and Amaroo at 3 d in anoxia. With 20 mol m–3 exogenous glucose, coleoptile extension and fresh weight increments in anoxic seedlings of IR22 were much closer to those in the two other genotypes, nevertheless protein concentration remained lowest on a fresh weight basis in the coleoptiles of IR22; indicating that protein synthesis has a lower priority for energy apportionment during anoxia than processes crucial to coleoptile extension. In contrast to these responses to anoxia imposed at imbibition, IR22 had nearly the same high tolerance to anoxia as Calrose and Amaroo, when anoxia was imposed on seedlings subsequent to 48 h aeration followed by 16 h hypoxic pretreatment. In fact, coleoptiles of anoxic IR22 had higher sugar concentrations and grew faster than Calrose, and exogenous glucose had no effect on the coleoptile extension of IR22. Excised coleoptile tips of IR22 and Amaroo with exogenous glucose had similar rates of ethanol production and were equally tolerant to anoxia. In conclusion, much of the anoxia ‘intolerance’ of IR22 when germinated in anoxia could be attributed to limited substrate availability to the embryo and coleoptile, presumably due to slow starch hydrolysis in the endosperm.
Key words: Anoxia, coleoptile, embryo, endosperm, ethanol production, germination, growth, Oryza sativa L., solute net uptake or loss, sugar availability.
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Introduction |
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Considerable variation exists among rice (Oryza sativa L.) genotypes in coleoptile extension during anoxia (Atwell et al., 1982; Setter et al., 1994), even though coleoptiles of rice seedlings are regarded as one of the most anoxia-tolerant plant tissues (Menegus et al., 1991; Pearce and Jackson, 1991). For example, the coleoptile extension of IR22 during anoxia was poor in comparison to other genotypes (Calrose: Atwell et al., 1982; Calrose and two other genotypes: Setter et al., 1994). Such differences might influence crop establishment, since extension of the coleoptile following the germination of seeds sown in flooded fields enables the seedlings to make contact with the atmosphere, thus gaining access to O2.
In rice coleoptiles, glycolysis linked to ethanol production is the predominant anaerobic pathway for energy production (Fan et al., 1997), as it is in many other plant species (ap Rees et al., 1987). The shoots of some rice genotypes have 1.4–1.7-fold faster rates of glycolysis in anoxia than in aeration (Gibbs and Greenway, 2003). With exogenous glucose, ethanol production rates in excised coleoptiles were stimulated by 33–59% in two anoxia-‘tolerant’ genotypes, but by as much as 3-fold in the anoxia-‘intolerant’ IR22 (Setter et al., 1994). Moreover, the analysis of concentrations of intermediates in glycolysis indicated insufficient substrate supply for glycolysis in IR22, but sufficient substrate in the more tolerant Calrose (Gibbs et al., 2000).
A gradual transition from normoxia via hypoxia to anoxia, rather than a sudden exposure to anoxia, may provide plants an opportunity for acclimation in many field situations (Drew, 1997). Hypoxic pretreatment might also improve the anoxia tolerance of IR22 seedlings, since extension rates of the coleoptile of intact seedlings following transfer from 3 d in hypoxia to anoxia were not affected, whereas coleoptile extension was very slow when seeds were germinated in anoxia (Atwell et al., 1982). Furthermore, the tolerance of rice seedlings to anoxia might be related to the developmental stage. For example, the early stage of coleoptile extension (2–5 mm) was most intolerant to anoxia (Atwell et al., 1982). After coleoptiles reached 4–5 mm, the extension rate was relatively insensitive to O2 supply, even in the anoxia ‘intolerant’ IR22 (Atwell et al., 1982). Developing tolerance was attributed to a shift from cell division to expansion (Atwell et al., 1982). Expansion mainly involves cell wall synthesis and solute uptake, both processes require less energy than protein synthesis (Penning de Vries, 1975) which would be high in dividing cells. However, in this paper, evidence is presented that the lesion in anoxic IR22 during germination is due to inadequate substrate mobilization from the endosperm. Once germination has progressed for 2 d in aeration and 16 h in hypoxia, this lesion was mainly relieved.
The experiments described in this paper evaluated anoxia tolerance of coleoptiles of intact seedlings of three rice genotypes at two growth stages, as affected by exogenous glucose. The genotypes were IR22 (previously reported as anoxia-‘intolerant’), Calrose (previously reported as anoxia-‘tolerant’) and Amaroo, the latter being derived from Calrose in selection trials in fields sown in standing water in NSW, Australia. The responses to anoxia of growth, tissue sugars, tissue K+ and its net loss, and soluble protein contents in coleoptiles of rice seedlings under two conditions were assessed. Firstly, seedlings were grown in anoxia from the start of imbibition. Secondly, seedlings were germinated under aeration for 2 d, followed by 16 h of hypoxic pretreatment, before exposure to anoxia. Furthermore, using excised, ‘healed’, hypoxically pretreated coleoptile tips with exogenous glucose, possible differences in energy production and ‘tissue tolerance’ to prolonged anoxia were assessed by measurements of rates of ethanol production and K+ and phosphate net uptake or loss.
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Preparation of the seedlings
Dehulled seeds of rice (Oryza sativa L. cv. IR22, Calrose, Amaroo) were surface-sterilized with acidic HgCl2 (0.1% w/v, in 0.1% HCl) for 3 min and then washed thoroughly with deionized water. Composition of the culture solution (mol m–3) was Ca2+, 0.5; Cl–, 0.6; morpholinoethanesulphonic acid (MES), 0.5; and 10 mg l–1 carbenicillin, with or without 20 mol m–3 glucose. The pH was adjusted to 6.5 using Ca(OH)2, contributing to the final Ca2+ concentration of 0.5 mol m–3. Culture vessels were surface-sterilized, solutions were autoclaved (before adding MES, glucose, or carbenicillin), and all procedures were performed in a laminar-flow hood to minimize numbers of micro-organisms. All experiments were conducted in the dark at 30 °C.
Experiments on tolerance to anoxia imposed since imbibition: comparison of IR22, Calrose and Amaroo
Twenty dehulled seeds of each genotype were transferred to conical flasks containing 200 ml of culture solution (composition as given above) without or with 20 mol m–3 glucose. Oxygen treatments were imposed prior to adding the seeds, by continuously bubbling each flask with humidified N2 gas or air. The mouth of each conical flask was sealed with two layers of Parafilm and wrapped tightly with two layers of aluminium foil. Oxygen in those solutions was not detectable by a Clark O2 electrode throughout the experiment. The samples were taken after 0, 1, 2, 3, and 6 d of anoxia and 1, 2, 3, and 4 d of aeration (see tables and figures). There were three replicates of each treatment for each sampling and genotype.
Growth of embryos was assessed by determination of fresh and dry weights. Growth of coleoptiles was evaluated as increments in lengths and fresh weights. Sugar, K+ and soluble protein contents and concentrations in endosperms, embryos, whole seeds, and coleoptiles were measured (methods are given below). Preliminary experiments showed that water loss during the 2 min of dissection of embryos from 10 seeds was less than 3% of the total fresh weight and there were no significant differences in sugar concentrations in embryos at 0, 5 and 10 min after excision (data not shown).
In another experiment designed to measure K+ and sugar net loss from seeds, a single seed was transferred into a tube containing 5 ml of anoxic or aerated culture solution flushed with N2 or air. Eight replicates of each genotype (IR22, Calrose and Amaroo) for each treatment were established. Solutions were collected and refreshed every 12 h for a period of 48 h. Solutions were stored at –20 °C prior to K+ and sugar measurements, taken only from those solutions in which the seed germinated (six to eight tubes for each treatmentx genotype combination).
Experiments with seeds germinated in aerated solution and then hypoxically pretreated prior to anoxia
Batches of 200 seeds of each genotype were transferred to individual PVC vessels containing 4.0 l of aerated culture solution. The seeds germinated and seedlings grew in the aerated solution (0.25 mol m–3 O2) for 48 h. The seedlings were then exposed to 16 h of hypoxic pretreatment (0.028 mol m–3 O2) before aerated or anoxic treatments were imposed.
Intact seedlings exposed to anoxia: comparison of IR22 and Calrose
After 16 h of hypoxic pretreatment, some seedlings of IR22 and Calrose were sampled for initial measurements of coleoptile lengths, fresh weights and sugar concentrations. The other seedlings were transferred to conical flasks containing 200 ml of anoxic culture solution with 10 mg l–1 carbenicillin, with or without 20 mol m–3 glucose. After 1 d and 3 d in anoxia, the seedlings were harvested to determine coleoptile extension, fresh weight increments and sugar concentrations. There were three replicates for each genotypexglucosexO2 treatment combination, with 20 seedlings per replicate flask.
Excised coleoptile tips exposed to anoxia: comparison of IR22 and Amaroo
Coleoptile tips of 7–10 mm were excised from hypoxically pretreated seedlings; such tips did not contain any leaf tissues. Excised coleoptile tips were transferred to Thunberg tubes containing hypoxic (0.028 mol m–3 O2) culture solution and treated as described in Colmer et al. (2001) and Huang et al. (2003). Excised coleoptile tips were maintained in hypoxic (0.028 mol m–3 O2) culture solution with 10 mg l–1 carbenicillin and 20 mol m–3 glucose for 5 h to enable wounds from excision to ‘heal’ (in previous publications this was termed ‘aged’) prior to aerated or anoxic treatments being imposed. Treatments were imposed by replacement of the solution in Thunberg tubes with 10 ml of anoxic nutrient solution or transfer of coleoptiles into conical flasks containing 50 ml of aerated nutrient solution. The solution composition was (mol m–3): K+ 0.25; Ca2+ 0.5; Mg2+ 0.1; NH4+ 0.10; NO3– 0.20; SO42– 0.505; H2PO4– 0.10, Fe-EDTA 0.0125 and (mmol m–3) Cl– 250; BO33– 6.25; Mn2+ 0.5; Zn2+ 0.5; Cu2+ 0.125; MoO42– 0.125; Ni2+ 0.25; and 0.5 mol m–3 MES (pH of complete solution was adjusted to 6.5 using Ca(OH)2), 10 mg l–1 carbenicillin, and 20 mol m–3 glucose. Excised coleoptile tips were exposed to anoxia for 120 h in experiments in which ethanol production was measured and 168 h in experiments on K+ and phosphate net uptake or loss. The solutions were refreshed and collected every 24 h (anoxic) or every 12 h (aerated). Vials containing deionized water in an ice bath connected to the outlet of the Thurberg tubes were used to trap ethanol in the gas stream. All samples were stored at –20 °C. The recovery of known ethanol standards from Thunberg tubes and vials after 24 h of flushing with N2 gas was 76%. The values of ethanol production corrected by this recovery value were reliable, but with higher standard errors when compared with values from later experiments with an improved trapping system in which 91% recovery was achieved. Bacterial numbers in nutrient solution after the final 24 h of anoxia were 1.7±0.13x106 ml–1. Petri dishes containing tryptic soy agar for bacteria counting with series dilution of nutrient solution were set in an anaerobic PVC vessel at 30 °C. The bacterial numbers in aerated nutrient solution after the final 12 h were 2.0±0.9x106 ml–1 (cultured on aerobic Petri dishes) and the total calculated fresh weight of bacteria in nutrient solution was less than 0.054% of the fresh weight of coleoptile tissues in the conical flasks (aerated treatment) and 0.013% in the Thunberg tubes (anoxic treatment).
Recovery of K+ and phosphate net uptake by excised coleoptile tips following 120 h of anoxic treatment was assessed. The nutrient solutions were sampled, refreshed and re-aerated. The solutions were then sampled and refreshed after 1, 2, 4, 6, and 8 h of re-aeration. After 8 h of re-aeration, the excised coleoptile tips were transferred to conical flasks with 50 ml aerated nutrient solution. The solutions were subsequently refreshed after another 16, 28 and 40 h. The solutions were stored at –20 °C prior to measurements of K+ and phosphate. There were three replicates of each genotypextreatment combination.
Analytical methods
Sugars in the various tissues were extracted in 80% (v/v) ethanol and determined as hexose units using anthrone (Yemm and Willis, 1954). The recovery of sugars during extraction was 98.6%. Starch in the rice coleoptiles was less than 5% of total sugars (expressed as hexose units; data not shown). Soluble proteins in embryos and coleoptiles were extracted with 50 mol m–3 K2HPO4/KH2PO4 (pH 7.4) and measured according to Bradford (1976). Ethanol in the nutrient solution and the traps was assayed using an enzymatic method (Beutler, 1983), and the concentration of ethanol in the nutrient solution never exceeded 1.2 mol m–3. Phosphate in the nutrient solution was measured using the molybdate and malachite green method as described by Motomizu et al. (1983). In all those assays, the same spectrophotometer (Shimadzu, UV-240, Tokyo, Japan) was used.
K+ in the various tissues dried at 70 °C for 2 d was extracted in 500 mol m–3 HCl for 2 d, as described by Hunt (1982). K+ in the nutrient solution and tissue extracts was measured using a flame photometer (Corning Medical and Scientific, Model 410, Cambridge, UK).
Statistical analysis of data
Analysis of variance for the various data sets used Genstat 4.2 software (5th edn, Lawes Agricultural Trust, Rothamsted Experimental Station).
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Experiment with seeds in anoxia from imbibition
Growth: Without exogenous glucose, growth of the coleoptiles of intact seedlings under anoxia was considerably slower in IR22 than for the other two genotypes (Table 1) even though growth in air was similar for all three genotypes (footnote of Table 1). This was already clear by 3 d after the start of imbibition for the comparison between Amaroo and IR22, with ratios of anoxia over air being 0.39 and 0.24 for extension and 0.14 and 0.09 for fresh weight for Amaroo and IR22, respectively. After 3 d in anoxia, Calrose was similar to IR22, but after 6 d in anoxia coleoptile growth in Calrose was greater than in IR22 (Table 1). Additionally, anoxia decreased the protein concentrations on a fresh weight basis by 58% in IR22, while this decrease was only 21% in Calrose and a mere 3% in Amaroo; comparing samples from seedlings after 6 d anoxia with those after 3 d in aeration (Table 2). Consistent differences were also found for embryo expansion; embryos of Amaroo under anoxia were already higher than IR22 in fresh weight at 2 d after imbibition, while any increase in size of the embryo of IR22 between 2 d and 3 d after imbibition had ceased altogether (Fig. 1).
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Anoxia tolerance of IR22 was improved by exogenous glucose (Tables 1, 2), yet at 6 d after imbibition in anoxia IR22 was still 34% lower in fresh weight (Table 1) and 48% lower in protein concentration than Amaroo (Table 2).
Sugar concentrations: Sugar concentrations in coleoptiles and seeds of IR22 were much lower than those in Calrose and Amaroo after 3 d and 6 d in anoxia (Fig. 2). Exogenous glucose increased the sugar concentrations in coleoptiles, but only slightly in seeds, of all three genotypes (Fig. 2).
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IR22 and Amaroo were selected to evaluate differences in sugar concentrations in embryos and endosperms during the early period of germination. For 3 d in anoxia, sugar concentrations on a water basis in the embryos and endosperms were much lower in IR22 than in Amaroo, and the values in Amaroo increased with time, while in IR22 the increase of sugar concentrations in the endosperms was quite small and sugar concentrations in the embryos decreased (Table 3). By contrast, after 3 d of anoxia the sugar accumulation ratio (embryo/endosperm) of IR22 and Amaroo were both 1.8 (Table 3), suggesting that sugar transport was more dependent on sugar concentrations in the endosperm than on the capacity for sugar uptake by the embryo. Higher sugar accumulation ratios in embryos of both genotypes after 1 d than after 3 d could be due to the high initial sugar concentrations in embryos (data not shown). With aeration, sugar concentrations in embryos of IR22 and Amaroo were similar, even though the values in the endosperm of IR22 were much lower than those of Amaroo (Table 3), albeit the concentrations in endosperm of aerated IR22 were much higher than in anoxic IR22 (Table 3). The sugar accumulation ratio (embryo/endosperm) of IR22 was higher in aeration than in anoxia at 2 d after imbibition (Table 3), indicating more energy-dependent sugar transport by the embryo in aeration than in anoxia. At day 3 in anoxia, sugar concentrations in the embryos in both IR22 and Amaroo were 1.7-fold greater than the concentrations in the coleoptiles, indicating a free energy gradient for transport (‘downhill’) from embryo to coleoptiles even under anoxia (calculated from Fig. 2 and Table 3).
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K+ net loss, K+ content and sugar net loss: During the first 12 h after imbibition both in anoxia or aeration, net K+ losses to the medium from seeds of IR22 were 4–5-fold higher than those from Amaroo, while net loss declined thereafter (Fig. 3). Substantial K+ loss to the medium from seeds of IR22 was confirmed by measurements of K+ contents in seeds (Fig. 4). The K+ content in embryos of both genotypes increased slightly, but the values in endosperms declined with time in both anoxia or aeration (Fig. 4). Furthermore, K+ depletion from endosperm of both genotypes during the second 24 h after germination was much higher in aeration than in anoxia (Fig. 4B).
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Sugar loss to the medium from seeds of IR22 was stable and never exceeded 27 nmol hexose seed–1 h–1 during the 48 h in anoxia (Fig. 3). On the other hand, sugar loss to the medium from seeds of Amaroo increased with time in anoxia and, during the last 12 h, reached nearly 3-fold the rate of loss from IR22 (Fig. 3). Higher sugar loss to the medium from seeds of Amaroo than IR22 was presumably related to the 3–4-fold higher sugar concentrations in both endosperm and embryo of Amaroo than IR22, over that period (data not shown).
Experiments in which anoxia was imposed after 48 h in aeration and 16 h in hypoxia
Intact seedlings—growth: The genotypic differences in anoxia tolerance were also tested for Calrose and IR22 seedlings after germination in aerated solution for 48 h and hypoxic pretreatment for 16 h. Under anoxia without exogenous glucose, the coleoptile length of IR22 increased by 29%, but there was no extension in Calrose (Fig. 5). Fresh weight increments of the coleoptiles of IR22 and Calrose seedlings during 3 d in anoxia were 0.97 and 0.77 mg fresh weight d–1, respectively (calculated from Fig. 5B). Twenty mol m–3 exogenous glucose stimulated coleoptile extension by 20% in Calrose, but not in IR22, between 1 and 3 d after anoxia was imposed (Fig. 5). However, exogenous glucose stimulated fresh weight increments of IR22, but not of Calrose (Fig. 5B), presumably much of this increase in fresh weight in IR22 was due to radial expansion of the coleoptile.
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Intact seedlings—sugar concentrations: Under anoxia, soluble sugar concentrations in the coleoptiles of both genotypes were similar and elevated by 20–30% with exogenous glucose (Fig. 6). In the seeds of anoxic seedlings, soluble sugar concentrations were 1.9–2.3-fold lower in IR22 than in Calrose (Fig. 6), but were 2-fold higher than those in seeds of IR22 at 6 d after seeds were imbibed in anoxia (Fig. 2). Exogenous glucose only slightly enhanced endogenous sugar concentrations in seeds of both genotypes (Fig. 6).
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Coleoptile tips—growth, soluble protein and sugar concentration: As shown above, there was much less difference between the genotypes in tolerance to anoxia when seedlings were first germinated in aeration followed by hypoxia prior to anoxia being imposed, than in seedlings grown in anoxia from imbibition. Possible difference in tissue tolerances to anoxia between coleoptiles of Amaroo and IR22 was tested using an excised system (coleoptile tissue only) supplied with 20 mol m–3 exogenous glucose. The excised coleoptile tips of Amaroo grew 1.5-fold and 1.9-fold faster than those of IR22 during anoxia and aeration, respectively (Table 4). The increase of fresh weight of coleoptile tips of both genotypes exposed to anoxia were reduced by about 70% compared with the aerated controls (Table 4), which was consistent with the results of intact and excised whole coleoptiles from rice seedlings of Calrose, with reductions of 70–80% due to anoxia (Huang et al., 2003). During recovery, the rates of fresh weight increment of coleoptile tips of Amaroo and IR22 were 1.2-fold and 1.5-fold higher than those in aeration for 120 h (Table 4).
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Soluble protein concentrations in excised coleoptile tips of both genotypes did not differ from the initial levels after 120 h in anoxia (Table 4). However, net protein synthesis of IR22 and Amaroo between 0 to 120 h of anoxia were 3.8 and 4.8 µg protein g–1 fresh weight h–1, compared with 28 and 23 µg protein g–1 fresh weight h–1 when aerated (calculated from Table 4 and the initial and final fresh weights of coleoptiles; not shown). Thus, rates of net protein synthesis in excised coleoptile tips in anoxia were 14% and 20% of those in aeration for IR22 and Amaroo, respectively.
The initial sugar concentrations in the excised coleoptile tips of Amaroo and IR22 were similar and had decreased by 24% after 120 h in anoxia (Table 4). The sugar concentrations in coleoptile tips of both genotypes in aerated solution for 120 h had increased by 1.6- to 1.7-fold (Table 4).
Coleoptile tips—ethanol production and net uptake of K+ and phosphate: During 120 h of anoxia, the rates of ethanol production on a fresh weight basis by excised coleoptile tips were the same for IR22 and Amaroo (Fig. 7A). In both genotypes, ethanol production rates were highest during the first 24 h of anoxia, and then decreased to about 6.5 µmol g–1 fresh weight h–1 for the remaining 96 h (Fig. 7A).
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During aeration, rates of K+ and phosphate net uptake by excised coleoptile tips of both genotypes were similar, with rates being 2.5–2.8-fold higher for K+ than for phosphate, and both declined with time (Fig. 7B, C). Rates of K+ and phosphate net uptake by anoxic coleoptile tips during 120 h of anoxia were between 0 and 0.09 µmol g–1 fresh weight h–1 for both genotypes (Fig. 7B, C). These average rates of net K+ and phosphate uptake by coleoptile tips of both genotypes during 120 h of anoxia were only 3% and 8% of those in aerated controls, respectively (Fig. 7B, C). During re-aeration, K+ net uptake by coleoptile tips of both genotypes increased rapidly and equally during the first 6 h and reached a peak after 6–8 h of re-aeration, declining thereafter (Fig. 7B). The rates of K+ net uptake at the peak, by coleoptiles of Amaroo and IR22 during re-aeration, were 90% and 85% of those during the initial 12 h in aeration (Fig. 7B). The phosphate net uptake rates recovered during 8 h of re-aeration and were maintained at high and relatively stable rates, during the next 40 h, at averages of 1.0 and 0.75 µmol g–1 fresh weight h–1 for Amaroo and IR22, respectively (Fig. 7C). The highest values for phosphate net uptake for Amaroo and IR22 during re-aeration were 73% and 90% of those during the initial 12 h in aeration (Fig. 7C).
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In earlier investigations using intact rice seedlings, IR22 was considered to be less anoxia-tolerant than Calrose (Atwell et al., 1982) and two other genotypes (Setter et al., 1994). The present study evaluated anoxia tolerance in coleoptiles of IR22, Calrose and Amaroo, with emphasis on the responses to exogenous glucose and growth stage. The response to substrate supply was evaluated since an earlier study indicated that the lower anoxia tolerance in IR22 may result from a limited supply of substrates for glycolysis (Gibbs et al., 2000). To test ‘tissue tolerance’ to anoxia of coleoptiles, as opposed to the response of the intact system, a system of excised coleoptile tips with exogenous glucose was developed. In this system, possible genotypic differences in response to anoxia were evaluated by assessment of the rates of ethanol production, net uptake or loss of solutes and net synthesis of soluble proteins. This system showed that differences in capacity for ethanol production and hence energy production in coleoptiles was not the cause of the genotypic difference in tolerance to anoxia.
Seeds germinated in anoxia
IR22 was much less anoxia-tolerant than Calrose, when anoxia was imposed at imbibition; findings which are consistent with the results presented in Atwell et al. (1982) and Setter et al. (1994). Amaroo was more anoxia-tolerant than Calrose, possibly because it was derived from Calrose in selection trials in fields in NSW Australia, where imbibed seeds were sown in standing water so that the young seedlings were submerged (L Lewin, personal communication). During such conditions, O2 deficiencies and hence anoxic zones in the seedlings are likely (Waters et al., 1989).
Twenty mol m–3 exogenous glucose improved the growth of coleoptiles in anoxic IR22 and to a lesser extent in Calrose, but there was no growth stimulation in Amaroo (Table 1). In earlier experiments, the growth of Calrose in anoxia did not respond to exogenous glucose (Atwell and Greenway, 1987), possibly because the exogenous glucose concentration used was only 5 rather than 20 mol m–3. In excised rice coleoptiles (Amaroo), there is about a 3-fold difference in rates of glucose net uptake between anoxic coleoptiles supplied with 5 or 20 mol m–3 glucose (S Huang, H Greenway, TD Colmer, unpublished data).
The substantial improvement in coleoptile growth of anoxic IR22 resulting from 20 mol m–3 exogenous glucose (Table 1) indicates that exogenous glucose overcame a substrate limitation. This hypothesis is further supported by the concomitant much larger increases in endogenous sugar concentrations upon supply of exogenous glucose in coleoptiles of IR22 than in the other two genotypes (Fig. 2). This limitation in substrate in coleoptiles of IR22 without exogenous glucose may be due to slow rates of sugar mobilization in the endosperm. Considering sugar transport, there was an ‘uphill’ concentration gradient between endosperm and embryo. However, there was no indication the genotypes differ in sugar transport, since the accumulation ratios of sugars in embryo versus endosperm were about 1.8 in both genotypes (after 3 d in anoxia; Table 3). It was concluded that there is a deficiency in carbohydrate mobilization in the endosperm of IR22 when germinated in anoxia. This deficiency being similar, though not as acute as, in anoxic seedlings of anoxia-intolerant cereals such as wheat and barley; the starch degrading enzyme 
-amylase, formed under anoxia in seeds of rice but not in wheat or barley (Perata et al., 1997). However, when seeds of IR22 were first aerated for 48 h and hypoxically pretreated for16 h, and then exposed to anoxia, the capacity for carbohydrate mobilization from endosperm was no longer limiting and differences between the genotypes in tolerance to anoxia became much less pronounced (Fig. 5). In aeration, sugar concentrations in the endosperm of IR22 were still lower than in Amaroo, but this was compensated for by a higher accumulation ratio (embryo/endosperm) in IR22 (Table 3). By contrast, under anoxia, the accumulation ratio of sugar in embryos of Amaroo and IR22 were both depressed to the same value, so the deficiency of sugars in the embryo and coleoptiles due to low sugar mobilization in the endosperm may have been exacerbated by limitations on sugar transport in anoxia (Table 3). Differences in response to anoxia between IR22 and Amaroo/Calrose, which depend on the germination stage, could be related to the expression of different -amylase isoforms during aeration and anoxia as reported by Hwang et al. (1999). Further studies are required on the regulation of mobilization of seed reserves in IR22 as compared with Amaroo, such as gene expression and activities of hydrolytic enzymes in different tissues in anoxia and aeration.
Exogenous glucose promoted the growth of IR22 in anoxia, which may have been due to increased energy availability since, in an earlier study, exogenous glucose increased ethanol production in excised anoxic rice coleoptiles (Setter et al., 1994). Exogenous glucose may also improve osmotic pressure for expanding cells, since sugars were one of the main solutes in anoxic rice shoots (Menegus et al., 1984) and coleoptiles (Atwell et al., 1982). Exogenous glucose would also provide a substrate for the synthesis of new cell wall polymers (Atwell and Greenway, 1987).
Genotypic differences in the responses of growth and soluble protein contents to exogenous glucose when germinating under anoxia (Table 5) are consistent with the hypothesis that apportionment of energy during an energy-deficit is regulated in tolerant tissues (Colmer et al., 2001). In the present case of IR22, increased glucose supply and, therefore, ethanol production and presumably energy supply, greatly increased coleoptile extension and fresh weight increments, while soluble protein accumulation was increased much less. An added experiment supplying 50 mol m–3 exogenous glucose showed a 42% increase in soluble protein content in coleoptiles of IR22 compared with 20 mol m–3 exogenous glucose, while the increase in extension of the coleoptiles was only 8% above that in seedlings with 20 mol m–3 glucose (data not shown). In the case of Amaroo, in which coleoptile length and fresh weight did not respond to exogenous glucose, there was still a stimulation of soluble protein net synthesis by 20 mol m–3 exogenous glucose. Energy apportionment to cell wall synthesis in preference to protein synthesis is an ‘energy efficient’ way to achieve coleoptile extension and hence increase the likelihood that the coleoptile might reach the atmosphere above flood waters before substrates are exhausted.
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Despite the large improvements in growth of the coleoptiles of anoxic IR22 elicited by exogenous glucose, fresh weight and soluble protein concentrations remained below those in anoxic Calrose and Amaroo (Tables 1, 2), although sugar concentrations in the coleoptiles of IR22 were highest (Fig. 2). Thus, restricted sugar availability to the coleoptiles might not be the only factor causing intolerance to anoxia in IR22. For example, there was more net K+ loss, presumably related to loss of membrane integrity in at least some cells, from seeds of IR22 than Calrose or Amaroo during the first days in both anoxia or aeration (Fig. 3). The net K+ loss to the medium, mainly from the endosperm (Fig. 4), has to pass through the aleurone layer, which is the only living tissue in endosperm (Sugimoto et al., 1998), indicating that cell injury in this layer may have occurred. The possible injury of cells in the aleurone layer in endosperms of IR22 during the early period of germination, may have, in turn, decreased the synthesis of hydrolytic enzymes. Perhaps this lesion was healed in aeration but not in anoxia, presumably due to much lower rates of energy production in anoxia than in aeration.
Seedlings hypoxically pretreated prior to anoxia
When germinated in aerated solution and hypoxically pretreated, there were no substantial differences in the responses to anoxia between seedlings of IR22 and Calrose (Fig. 5). Nevertheless, exogenous glucose during anoxia stimulated coleoptile extension of Calrose and fresh weight increment of IR22 (Fig. 5). Consistently, after 3 d at 0.0625 mol m–3 O2, coleoptile extension was not slowed during subsequent anoxia in either IR22 or Calrose (Atwell et al., 1982). The increments in lengths and fresh weights presented by Setter et al. (1994) also showed little difference between IR22 and Calrose during 5 d of anoxia following an initial 4 d in aeration. However, in the present study, sugar concentrations were 82–95 µmol hexose g–1 fresh weight in coleoptiles of IR22 and Calrose after 1 d and 3 d in anoxia (Fig. 6), while in the experiments by Setter et al. (1994) sugar levels in Calrose and IR22 were only 25–55 and 2–37 µmol hexose g–1 fresh weight, respectively. One reason for this discrepancy might be an interaction between anoxia and growth cessation and/or senescence of the coleoptile, since leaf development would already have been substantial when seedlings had been aerated for 4 d before imposing anoxia, as described in the experiments by Setter et al. (1994). That senescence develops in coleoptiles of aerated seedlings at 2–3 d after imbibition is supported by (a) decreases in soluble protein concentrations of coleoptiles between 3 d and 4 d after germination in aerated seedlings (Table 2), (b) cessation of coleoptile extension after 3 d in aeration (S Huang, H Greenway, TD Colmer, unpublished data), and (c) dramatic decreases in total adenine nucleotide concentrations in rice coleoptiles later than 2 d after the start of imbibition in aerated conditions (Ishizawa et al., 1999). A second reason could be that the hypoxic pretreatment of the seedlings in the present study and those in the experiments of Atwell et al. (1982) may have resulted in a higher ‘anoxia-tolerance’ than for the anoxically-shocked seedlings of Setter et al. (1994).
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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The improvement of anoxia tolerance in intact seedlings of IR22 by exogenous glucose when anoxia was imposed at imbibition, demonstrated that intolerance of IR22 was due to slow sugar mobilization and not to lesions in the metabolic machinery for glycolysis and ethanol production. Experiments with excised coleoptile tips incubated in nutrient solution with 20 mol m–3 exogenous glucose demonstrated that tissues from IR22 and Amaroo had similar rates of ethanol production during 120 h of anoxia (Fig. 7A), supporting the hypothesis by Gibbs et al. (2000) that there is no lesion in capacity of ethanol production in IR22, provided sufficient substrates are available. Furthermore, coleoptile tips from both genotypes supplied with exogenous glucose were equally tolerant to anoxia as supported by (i) no net loss of K+ or phosphate during 168 h of anoxia, and (ii) equally rapid recovery of K+ and phosphate net uptake during re-aeration after 120 h of anoxia (Fig. 7B, C). Whether differences in anoxia tolerance at the seedling stage among other genotypes of rice are also related to the mobilization of substrate, and/or metabolism in the coleoptiles, requires further study.
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Acknowledgements |
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Dr Tim Setter and Professor Pierdomenico Perata gave constructive comments on an early draft of this manuscript. Dr Laurie Lewin, from Yanco Agricultural Institute NSW, provided rice seeds and information on aspects of the genotypes used. Dr Brian Atwell gave useful suggestions for improving the manuscript. SH is grateful to the UWA for a University Postgraduate Award and International Postgraduate Fee Waiver Scholarship.
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