Annals of Botany 91: 271-278, 2002
© 2002 Annals of Botany Company
Responses by Coleoptiles of Intact Rice Seedlings to Anoxia: K+ Net Uptake from the External Solution and Translocation from the Caryopses
1 School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, 6009 WA, Australia
* For correspondence: Tel +61 8 9380 1993, Fax +61 8 9380 1108, e-mail tdcolmer{at}cyllene.uwa.edu.au
Received: 6 August 2001; Returned for revision: 20 November 2001; Accepted: 6 February 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONLITERATURE CITED |
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This study evaluated the effects of anoxia on K+ uptake and translocation in 3–4-d-old, intact, rice seedlings (Oryza sativa L. cv. Calrose). Rates of net K+ uptake from the medium over 24 h by coleoptiles of anoxic seedlings were inhibited by 83–91 %, when compared with rates in aerated seedlings. Similar uptake rates, and degree of inhibition due to anoxia, were found for Rb+ when supplied over 1·5–2 h, starting 22 h after imposing anoxia. The Rb+ uptake indicated that intact coleoptiles take up ions directly from the external solution. Monovalent cation (K+ and Rb+) net uptake from the solution was inhibited by anoxia to the same degree for the coleoptiles of intact seedlings and for coleoptiles excised, ‘aged’, and supplied with exogenous glucose. Transport of endogenous K+ from caryopses to coleoptiles was inhibited less by anoxia than net K+ uptake from the solution, the inhibition being 55 % rather than 87 %. Despite these inhibitions, osmotic pressures of sap (
sap) expressed from coleoptiles of seedlings exposed to 48 h of anoxia, with or without exogenous K+, were 0·66 ± 0·03 MPa; however, the contributions of K+ to sap were 23 and 16 %, respectively. After 24 h of anoxia, the K+ concentrations in the basal 10 mm of the coleoptiles of seedlings with or without exogenous K+, were similar to those in aerated seedlings with exogenous K+. In contrast, K+ concentrations had decreased in aerated seedlings without exogenous K+, presumably due to ‘dilution’ by growth; fresh weight gains of the coleoptile being 3·6- to 4·7-fold greater in aerated than in anoxic seedlings. Deposition rates of K+ along the axes of the coleoptiles were calculated for the anoxic seedlings only, for which we assessed the elongation zone to be only the basal 4 mm. K+ deposition in the basal 6 mm was similar for seedlings with or without exogenous K+, at 0·6–0·87 µmol g–1 f. wt h–1. Deposition rates in zones above 6 mm from the base were greater for seedlings with, than without, exogenous K+; the latter were sometimes negative. We conclude that for the coleoptiles of rice seedlings, anoxia inhibits net K+ uptake from the external solution to a much larger extent than K+ translocation from the caryopses. Furthermore, K+ concentrations in the elongation zone of the coleoptiles of anoxic seedlings were maintained to a remarkable degree, contributing to maintenance of sap in cells of these elongating tissues.
Key words: Anoxia, caryopsis, coleoptile, Oryza sativa, osmotic pressure, potassium, uptake, translocation, seedlings, spatial distribution.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONLITERATURE CITED |
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Coleoptiles of intact rice seedlings are one of the few plant organs that elongate during anoxia (Atwell et al., 1982; Alpi and Beevers, 1983; Menegus et al., 1984; Pearce and Jackson 1991; Setter and Ella, 1994). In an experiment with 3–4-d-old rice seedlings, increases in fresh weight under anoxia were 16-fold greater for the coleoptiles than for the leaves inside the coleoptiles (Menegus et al., 1984). Elongation of the coleoptile of intact seedlings was faster under anoxia, than in aerated conditions, during investigations by Alpi and Beevers (1983) and Menegus et al. (1984). Yet, in other studies, elongation rates during anoxia were at best equal to those in aerated conditions (Atwell et al., 1982; Pearce and Jackson, 1991). Coleoptile elongation during anoxia is of adaptive significance since contact of the seedling with the atmosphere can be established, hence providing an O2 source to submerged seedlings (Kordan, 1974).
In the coleoptile of rice, glycolysis linked to ethanolic fermentation is the predominant anaerobic pathway of sugar catabolism (Bertani et al., 1980; Menegus et al., 1991), as it is in other species (ap Rees et al., 1987), thus providing at least some ATP production during anoxia. The data in Alpi and Beevers (1983) provide an indication of the priority of various growth processes in coleoptiles of rice when energy supply is severely limited; elongation rates increased, while dry weight and protein increments were decreased. Cell expansion still requires some ATP, e.g. energy-dependent net uptake of solutes is required to maintain cell turgor. K+ and sugars are major solutes contributing to the osmotic pressure of the cell sap (sap) in the coleoptile of rice seedlings (Atwell et al., 1982; Menegus et al., 1984), and amino acids also contribute to sap, particularly during exposure to anoxia (Menegus et al., 1984). In addition, cells in the coleoptiles of anoxic rice seedlings may well take up K+, since their membrane potential remains more negative than the K+ diffusion potential (Zhang and Greenway, 1995). However, net K+ uptake by excised rice coleoptiles was 12–25 times lower in anoxia, than in aerated solution (Colmer et al., 2001).
The experiments described in the present paper evaluated net K+ uptake, and K+ translocation from the caryopses to the coleoptiles, in intact seedlings of rice (Oryza sativa L. cv. Calrose). Aspects studied were the effects of anoxia and exogenous K+ supply, at 0·25 mol m–3, on growth, tissue K+ concentrations, the contributions of K+ to sap, and the spatial distributions of K+ concentrations and deposition rates along the axes of the coleoptiles. Rb+ uptake over 1·5–2 h was used to evaluate whether K+ from the external solution was taken up directly by intact coleoptiles. Ethanol synthesis and net K+ uptake have been studied in the past using excised coleoptiles (e.g. Gibbs et al., 2000; Colmer et al., 2001). Responses to anoxia of coleoptiles of intact seedlings (present study) were compared with those of excised coleoptiles (data from present study and Colmer et al., 2001).
Validation of the excised system is an important issue, since many experiments require excision to allow interpretation. Experiments on ethanolic fermentation, for example, are almost impossible to interpret if measurements are made using intact seedlings, since one cannot distinguish between ethanol produced by caryopses or by coleoptiles. Moreover, studies on the mechanisms of K+ uptake by the coleoptile from the medium are also confounded in intact seedlings, since K+ can flow to the coleoptiles from endogenous pools in the caryopses and roots, or from that recently absorbed from the medium by these organs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONLITERATURE CITED |
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Preparation of seedlings
Dehulled seeds of rice (Oryza sativa L. cv. Calrose) were surface sterilized with acidic HgCl2 (0·1 % w/v, in 0·1 % HCl) for 3 min (Atwell et al., 1982) and then washed thoroughly with deionized water. Batches of 200 seeds were transferred to a mesh screen submerged within a PVC vessel containing 4 l of aerated culture solution. The composition of the solution was (mol m–3): Ca2+, 0·5; Cl–, 0·6; MES, 0·5; the pH had been adjusted to 6·5 using Ca(OH)2. The culture vessels were surface sterilized, solutions were autoclaved (before adding MES), and all procedures were performed in a lamina flow hood. Each culture vessel had a gas-tight lid with a small inlet and outlet for gas lines. The growth of seedlings, and all the experiments, were conducted in the dark at 30 °C.
Experiments with intact seedlings
The seeds germinated and grew in aerated solution (containing 0·25 mol m–3 O2) for 48 h. The seedlings were then exposed to a 16-h hypoxic pretreatment (0·028 mol m–3 O2). Subsequently, each mesh screen holding a group of seedlings was transferred to a new PVC vessel containing 4 l of aerated or anoxic culture solution (composition given above), which also contained either 0 or 0·25 mol m–3 KCl. A hypoxic pretreatment was used so as to avoid anoxic shock and, in addition, hypoxia promoted coleoptile rather than leaf growth (own observations). At the time of imposing treatments, the seedlings raised as described above had a coleoptile of about 20 mm, but some leaf tissues were enclosed; these reached about 40–50 % of the length of the coleoptile. Despite the presence of these leaf tissues, we refer to the samples of coleoptiles with enclosed leaves as ‘coleoptiles’. The concentration of K+ used was based on preliminary experiments with external K+ between 0·1 and 0·4 mol m–3, in which rates of net K+ uptake by excised coleoptiles in aerated solution were saturated at 0·2 mol m–3. There were three replicate vessels for each treatment.
After the 16-h hypoxic pretreatment, some coleoptiles were sampled, for initial measurements of length, fresh weights, and concentrations of sugars and K+. After 24 h of treatments, intact seedlings were ‘washed’ (3 x 3 min) in culture solutions without K+, while maintaining either aeration or anoxia. Coleoptile lengths and fresh weights were then measured, and tissues were processed for measurements of K+ concentrations. Coleoptiles of seedlings exposed to anoxia for 48 h were harvested for sap measurements.
In a second experiment, intact seedlings were raised and treated as described above. Coleoptiles were then ‘washed’ and cut into 2 mm segments using razor blades held in a rack, for measurements of the spatial distribution of K+ along the axes of the coleoptiles. The elongation zone of coleoptiles of seedlings in anoxic solution was also assessed by marking coleoptiles with small vertical lines at various intervals using a fine pen containing xylene-free ink, and then observing the patterns of ink displacements after 24 h of treatment. Each replicate (three per treatment) consisted of a batch of seedlings, each in a separate 4-l vessel.
In a third experiment, intact seedlings were raised and treated as described above. Short-term Rb+ uptake from the medium was assessed between 22 and 24 h after the O2 and K+ treatments were imposed. KCl in the medium was replaced by RbCl at 0·25 mol m–3 for the final 1·5 h (aerated) or 2 h (anoxic) of the treatments. The seedlings were then ‘washed’ with culture solution without Rb+ or K+ for 3 x 3 min, coleoptiles were excised, and 2-mm segments were harvested for subsequent analysis of Rb+. Each replicate (two per treatment) consisted of a batch of seedlings, each in a separate 4-l vessel.
Experiments with excised coleoptiles
Seedlings were raised in aerated culture solution and given the hypoxic pretreatment as described above, after which the coleoptiles were excised. Batches of coleoptiles were transferred to ‘Thunberg tubes’ (anoxic treatments) or small conical flasks (aerated treatments) and treated as described in Colmer et al. (2001). In brief, each tube contained 10 ml, and each flask contained 100 ml, of solution (same composition as the culture solution given above plus 20 mol m–3 glucose and 10 g m–3 carbenicillin). Excised coleoptiles were ‘aged’ for 5 h in hypoxic (0·028 mol m–3 O2) solution prior to measurements of rates of net K+ uptake, determined by depletion from the external solution with an initial K+ concentration of 0·25 mol m–3 (depletion was 10–20 %).
Analytical methods
Tissues were oven dried at 70 °C for 2 d, after which K+ and Rb+ were extracted by shaking in 500 mol m–3 HCl for 2 d (Hunt, 1982) at room temperature. K+ and Rb+ in appropriate dilutions of the extracts were measured using a flame photometer (Model 410; Corning Medical and Scientific, Cambridge, UK) or an atomic absorption spectrometer (Analyst 300; Perkin Elmer Instruments, Norwalk, CT, USA).
Osmotic pressure of sap (sap) expressed from freeze/thawed coleoptiles was measured using a freezing point depression osmometer (Model ONE-TEN, Fiske Associates, Norwood, MA, USA). Coleoptiles were blotted to remove surface solution, sealed in cryo-vials, and frozen in liquid N2. Samples were thawed while still in their sealed vials and then crushed in a stainless-steel press to extrude sap, which was immediately analysed.
Soluble sugars (hexose units) in samples of coleoptiles frozen in liquid N2 and freeze-dried, were extracted in 80 % (v/v) ethanol and determined using anthrone (Yemm and Willis, 1954).
Calculations
Rates of K+ translocation from caryopses to coleoptiles (µmol g–1 f. wt h–1) were calculated from the data on seedlings without exogenous K+ (Table 1), using:
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(WII x CII – WI x CI)/[(WII +WI) x t x 0·5)](1)
where W = fresh weight of coleoptile, C = concentration, subscripts I and II denote samples taken at two times, and t = time between samplings (h); in the present study t = 24 h.
Analyses of spatial patterns of deposition rates of materials along growing plant organs were pioneered by Silk and Erickson (1979). We do not have data on spatial patterns of extension within the growth zone for the coleoptile of rice, so could not use the equations provided by Silk et al. (1984); instead we used a simpler approach to estimate rates of K+ deposition (µmol g–1 f. wt h–1) along the coleoptile of anoxic rice seedlings (see below), using the following parameters: length of the elongation zone, growth, and spatial patterns of K+ contents at two times.
To determine the deposition rates between times 1 and 2, the following are needed: (a) the locations of the original boundaries of each segment (time 1; initial sampling); (b) the new locations of these original boundaries (time 2; top and bottom boundary of each segment, calculated using eqns 2 and 3); (c) the K+ contents and fresh weights along the coleoptile per segment cut from the coleoptiles at the two sampling times.
The original boundaries of the segments would have shifted to new locations, in millimetres from the coleoptile base, as given by the following equations for:
(a) segments that at time 1 were in the elongation zone:
topn = n(1 + E)
(2a)
andbottomn = (n – 1)(1 + E)(2b)
where E is the elongation factor = 
L/Le, in which L is the increase in length of the coleoptile and Le is the length of the elongation zone, is the length of the segments at time 1, in our case all 2 mm, and n defines the segment number, being 1 for the most basal 2 mm, 2 for the next, and so on.
(b) segments that were above the elongation zone at time 1 (i.e. n > Le/). Displacement of both the top and bottom boundary of each segment will be L, so that new locations of the tops of these segments are:
topn = (n) + L(3)
(for n > Le/); the terms are as defined above.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONLITERATURE CITED |
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Ageing requirements after excision
For comparisons between intact and excised coleoptiles it is crucial to ‘age’ after excision, in order to heal any wound injury. This is particularly important in studies of plant responses to anoxia, since Butler et al. (1990) showed wound response enzymes were not induced if potato tubers, an anoxia-intolerant tissue, were injured and then exposed to O2 deficiency. For aerated maize roots, Gronewald et al. (1979) assessed that 4 h was required to repair injury, due to excision. During the present work, rice coleoptiles showed a net K+ loss at 1·5 µmol g–1 f. wt h–1 during the first hour after excision, decreasing to 0·16 µmol g–1 f. wt h–1 by the second hour, and losing no K+ during the third hour. In excised rice coleoptiles ‘aged’ for 5 h in hypoxic solution with 20 mol m–3 glucose, vigorous net uptake of K+ occurred when K+ was added to the external solution. Thus, 5 h of ‘ageing’ in hypoxic solution was adopted in all our experiments with excised coleoptiles.
Growth and net K+ uptake
In aerated solution, the coleoptiles of intact seedlings gained fresh and dry weight faster when exogenous K+ was supplied (Table 1). In anoxia, exogenous K+ affected neither fresh weight gains (Table 1) nor elongation rates (data not shown); the fresh weight gains during anoxia were only 21–28 % of the gains for coleoptiles of seedlings in aerated solution. In contrast, the fresh weight gains of coleoptiles of 3-d-old intact rice seedlings exposed to anoxia for 24 h was 61 % of those of aerated seedlings (Menegus et al., 1984). Menegus et al. (1984) separated the leaves from coleoptiles, while we did not (cf. Materials and Methods); so the larger growth reductions in our study could be due to the larger inhibition by anoxia on leaf growth, rather than on coleoptile growth, i.e. the aerated controls in our study presumably had considerable leaf growth inside the coleoptile. Root growth was substantial in aerated seedlings, but at most slight in anoxic seedlings (Table 1).
Comparing growth of the intact coleoptiles with that of excised coleoptiles; fresh weight increments in coleoptiles of aerated seedlings were 2·4 times greater than in excised coleoptiles with 20 mol m–3 exogenous glucose. Anoxia inhibited growth by 70 and 90 % in the intact and excised coleoptiles, respectively (data not shown).
In aerated solutions, Rb+ or K+ uptake rates from the solution were substantial and similar for intact and excised coleoptiles (Table 2). Furthermore, Rb+ uptake by the rice coleoptiles was similar to rates observed for excised tomato and maize roots and 400 µm wide slices from maize leaves (Table 2). The hydrophobic cuticle impedes ion uptake by leaf tissues, hence narrow slices are usually required to measure ion uptake and loss by leaf tissues (Jeschke, 1976). However, the surface of the rice coleoptiles clearly did not pose a substantial barrier to K+ or Rb+ uptake. Presumably, the cuticle development in coleoptiles grown submerged was less than that in leaf surfaces exposed to the atmosphere, allowing the coleoptile of a submerged rice seedling to absorb solutes from the external solution.
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K+ concentrations in the coleoptiles were highest for seedlings aerated with K+ > anoxic with K+ > anoxic without K+ > aerated without K+ (Table 1). K+ content per coleoptile increased over the 24 h of anoxia (Table 1); in the seedlings without exogenous K+, this K+ was presumably derived from the caryopses. A balance sheet of changes in the K+ content of coleoptiles, caryopses and roots showed that under anoxia there was no substantial net K+ leakage from the intact rice seedlings to the solution without exogenous K+, and there was net uptake by seedlings supplied with exogenous K+. However, in the roots of anoxic seedlings, K+ content had decreased by 5 % and 14 % after 24 h for those seedlings with or without exogenous K+, respectively (Table 1). The declines in K+ content in anoxic rice roots with exogenous K+ was much smaller than the 30 % net loss of K+ during 24 h of anoxia from hypoxically pretreated roots of intact wheat plants in nutrient solution with K+ at 2·4 mol m–3 (Greenway et al., 1992). In contrast, Reggiani et al. (1993) reported accumulation of K+ by roots and shoots of 4-d-old anoxically shocked wheat seedlings with shoots in the anoxic gas phase. However, in the experiments by Reggiani et al. (1993) exogenous K+ was 30–60 mol m–3, so that uptake of K+ would not require energy. This view is supported by the conclusion by Trought and Drew (1980) that transport of ions to the shoots of 13–17-d-old wheat with roots in anaerobic solution moved by mass flow across damaged root tissues.
Anoxia inhibited K+ or Rb+ uptake from the solution by 83–91 %, in coleoptiles of either intact seedlings or when excised (Table 2). For the intact seedlings, uptake rates by the coleoptiles directly from the solution were obtained by three methods which all gave similar results. The methods were: (a) as differences between seedlings with or without exogenous K+ (calculated from Table 1), (b) from the differences between the increases in K+ content of the coleoptiles (Table 1) and decreases in K+ content of the caryopses (data not shown), and (iii) supplying Rb+ for 1·5–2 h (Table 2). The first two assessments were taken from uptakes over 24 h anoxia and hence may have included K+ uptake from the solution by the small root system and/or caryopses, and this K+ may have contributed to K+ translocation to the coleoptile. However, the short-term Rb+ uptake provides a reasonable assessment of the Rb+ taken up directly by the coleoptile from the external solution, though 1·5–2 h is, of course, too long to measure unidirectional influx.
The severe inhibition due to anoxia of K+ and Rb+ uptake from the solution, contrasts with the much milder inhibition of 55 % for translocation of endogenous K+ from the caryopses to the coleoptiles (calculated from the treatments without exogenous K+; Table 1), i.e. phloem transport was inhibited less by anoxia than net uptake of exogenous K+. An alternative to K+ transport via the phloem would be K+ leakage from caryopses and roots to the solution, followed by re-absorption by the coleoptiles. However, this notion is unlikely, because if the K+ had leaked to the solution, concentrations in the medium without exogenous K+ would have remained extremely low, reaching at most 10 mmol m–3 at the end of the experiment (calculated from the K+ decreases in the caryopses and the volume of nutrient solution).
Contribution of K+ to sap
After 48 h of treatments, osmotic pressure of sap (sap) expressed from coleoptiles was 0·66 ± 0·03 MPa for anoxic seedlings with or without exogenous K+, which was similar to the higher range of sap reported for rice coleoptiles by Atwell et al. (1982). The contributions of K+ to sap of the coleoptiles were 23 and 16 %, for anoxic seedlings with or without exogenous K+, respectively. These values were lower than the 33 % contribution of K+ to sap in 3-d-old rice coleoptiles after 48 h in anoxia (Menegus et al., 1984). Low K+ contributions would be partly compensated for by increases in amino acids, after 48 h anoxia contributed 22 % to the sap in shoots of intact rice seedlings (Menegus et al., 1984), while alanine contributed 52 % to the total amino acids (Menegus et al., 1993). Sugars are another important contributor to sap in the coleoptile of rice seedlings (Atwell et al., 1982; Menegus et al., 1984). In the coleoptile of intact seedlings grown in the present study, soluble sugars decreased during 24 h of anoxia, from 99·1 ± 16·1 to 83·1 ± 9·6 µmol hexose units g–1 f. wt, the latter value would still contribute about 0·23 MPa to sap.
Sugar sequestration in osmotic pools, especially in the vacuole, could possibly compete with other processes such as anaerobic catabolism and synthesis of new cell wall materials. In coleoptiles of anoxic rice seedlings in the present experiments, the amount of sugar flowing to the osmotic pool over the 24 h period was 0·47 µmol g–1 f. wt h–1 (calculated from Table 1 and the sugar data in the preceding paragraph); this was only 12 % of the amount of sugar flowing to ethanolic fermentation in excised rice coleoptiles over the same time period (Colmer et al., 2001). The diversion of sugars to osmotic pools, and to any possible cell wall synthesis, would have repercussion only when substrates for glycolysis were limiting. In that case, synthesis of alanine rather than ethanol may be important, since ethanol leaks to the external solution, whereas alanine does not. Since 2 mol of alanine are derived per mol of glucose, alanine synthesis would contribute to any compensation for decreases in contributions of sugars to osmotic pressure.
Spatial distribution of K+ along the coleoptile
For an elongating organ like a coleoptile it was of interest to determine spatial patterns of K+ concentrations and net uptake rates along the coleoptile; in particular, since in rice coleoptiles ethanolic fermentation rates on a fresh weight basis were three times faster in the basal 3 mm than in the 11–14 mm tip (Setter and Ella, 1994).
K+ concentrations in each 2 mm segment are the end result of increases due to net K+ uptake and decreases due to volume expansion. Coleoptiles elongated and expanded, in both dimensions, growth being much larger when the seedlings were in aerated than in anoxic solutions (cf. initial against final values in Fig. 1A). The elongation in anoxic coleoptiles occurred in the basal 0–4 mm, with a total extension of 1 mm over 24 h in anoxia. There was no elongation of tissues further towards the coleoptile tip (data not shown). Much of the volume increase in the aerated seedlings was presumably associated with development of leaves rather than being expansion of coleoptiles. In anoxia the radial increases in volume of the 4 mm growing zone were about 17 %. This compares with a contribution of 13 % by leaves to the total growth of anoxic rice shoots, determined by separate sampling of coleoptiles and leaves in a similar experiment with intact rice seedlings (Menegus et al., 1984). So, most of the radial expansion in the coleoptiles of anoxic seedlings in the present experiment was presumably due to volume increases by the coleoptile itself.
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Over the 24 h of the experiment, increases in K+ content per unit length were predictably large in coleoptiles of aerated seedlings with exogenous K+, but were only slight in the coleoptiles of anoxic seedlings (Fig. 1B). K+ concentrations changed very little in the basal 10 mm of coleoptiles of seedlings with exogenous K+ (anoxic or aerated), or in those of anoxic seedlings without exogenous K+ (Fig. 1C). Segments above 10 mm in the coleoptile of aerated seedlings with exogenous K+ reached higher K+ concentrations than in the basal zones (Fig. 1C). In contrast, in anoxic coleoptiles, the tissues above 10 mm decreased to lower K+ concentrations than at the start of anoxia, particularly in the seedlings without exogenous K+. Yet, these K+ concentrations remained above those in the aerated seedlings without K+ (Fig. 1C), because ‘dilution’ due to growth would have been larger in the coleoptiles of aerated, than of anoxic, seedlings (Fig. 1A and Table 1).
The data in Fig. 1 and our assessment of the 4 mm length of the elongation zone enable evaluation of rates of K+ deposition (µmol g–1 f. wt h–1) for the different segments along the coleoptiles of seedlings. These rates were only calculated for the anoxic seedlings (Fig. 2). K+ deposition rates in the basal 6 mm were nearly independent of the presence or absence of exogenous K+; the rates were 0·6–0·87 µmol g–1 f. wt h–1 (Fig. 2). The deposition rates estimated for the basal zones are likely to be underestimates, since uptake of K+ by leaf tissues of 3–4-d-old anoxic rice was negligible (Menegus et al., 1984). Above 6 mm from the base, deposition rates were always lower for anoxic seedlings without exogenous K+, compared with those with exogenous K+ (Fig. 2). In the absence of exogenous K+, the K+ deposited in the basal 6 mm must have been derived mainly from the caryopses; however, there was also a possible contribution from other tissues along the coleoptile, since tissues at some positions showed net remobilization of K+ (i.e. negative rates of deposition). The patterns of deposition indicate that as endogenous K+ concentrations became low, there was a preferential flow of K+ to the basal tissues of the coleoptile. This maintained K+ concentrations in the basal zones to a remarkable degree, and is consistent with the higher energy supply, predicted from the higher rates of ethanolic fermentation in the basal than in the tip tissues of rice coleoptiles (Setter and Ella, 1994). It remains possible that elongation rates of cells along the axes of the coleoptiles may differ even within this basal 4 mm zone, as has been observed for the ‘growth zone’ in maize root tips (Silk et al., 1984) and in sorghum leaves (Bernstein et al., 1993). Thus, the K+ deposition rates at some locations within the basal 4 mm of the coleoptile of anoxic rice seedlings could be even higher than presented in Fig. 2.
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The evidence described above suggests that the demand for K+ in cells at different positions along the coleoptile of rice may differ considerably; such differences may, at least partly, be satisfied by spatial differences in rates of K+ uptake directly from the external solution. Hence, spatial patterns of Rb+ uptake along the coleoptiles of intact seedlings were also determined. Coleoptiles in this experiment were about 12 mm long, and 22 h after start of anoxia KCl was replaced by RbCl, for the final 1·5 h (aerated) or 2 h (anoxic). The coleoptiles of aerated seedlings showed highest rates of Rb+ uptake by the tissues near the tip, while rates in the most basal 2 mm were also faster than in the middle 2–10 mm (Fig. 3). Inhibition of Rb+ uptake due to anoxia was about 90 % throughout the basal 10 mm. Thus, Rb+ uptake from the solution by the cells in the basal elongation zones was as sensitive to anoxia as for cells in the non-elongating zones. This pattern contrasted with the higher rates of deposition of endogenous K+ in the basal tissues than in the tissues nearer the tip (Fig. 2), indicating transport from the caryopses contributes substantially to the K+ deposition in these elongating zones.
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GENERAL DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONGENERAL DISCUSSIONLITERATURE CITED |
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Comparison between solute uptake by coleoptiles from solution and via translocation from caryopses
The 83–91 % inhibition of K+ and Rb+ uptake due to anoxia in coleoptiles of intact rice seedlings is consistent with previous findings for excised coleoptiles (Colmer et al., 2001). This contrasted with the much less severe inhibition of 55 % for K+ translocation from the caryopses, via the phloem, to the coleoptiles. Even more impressively, there was no inhibition of K+ transport at all over 24 h from the caryopses to the coleoptiles in anoxic 3-d-old intact rice seedlings in an earlier study (Menegus et al., 1984). The capacity for transport of K+ from the caryopses, to the coleoptiles, in anoxic seedlings may be even greater than indicated above, because the transport might be limited by the slower development of the sink (i.e. slower growth rate of the coleoptile in anoxic than in aerated solution), rather than by an inhibitory effect of anoxia on the capacity of the transport system. Translocation of K+ contained in the caryopses is important under anoxia, as shown by comparing the net uptake rates for the coleoptiles of intact seedlings without or with exogenous K+, being 0·76 and 1·28 µmol g–1 f. wt h–1, respectively (calculated from Table 1). An almost identical rate of transport from the caryopses to the coleoptiles of rice seedlings in deionized water occurred in the experiment by Menegus et al. (1984). Thus, translocation of endogenous K+ from the caryopses contributed more to K+ intake by the coleoptiles than did net K+ uptake from the external solution with K+ at 0·25 mol m–3.
Sugar translocation from caryopses to coleoptiles of rice seedlings also remained operational during anoxia, thus sustaining rapid ethanolic fermentation. This sugar translocation is shown by the soluble sugar concentration in rice coleoptiles during 24 h of anoxia, which had only decreased by 16 %, while the fresh weight of the coleoptiles increased by 34 %. Ethanolic fermentation can be estimated from the data earlier obtained with an excised system (Colmer et al., 2001); being 5·1–7·5 µmol g–1 f. wt h–1 during 60 h of anoxia. Furthermore, net glucose uptake was 2·6 µmol g–1 f. wt h–1 during 48 h of anoxia (Colmer et al., 2001), so in the intact seedlings sucrose transport to the coleoptiles would have been at least 1·3 µmol g–1 f. wt of coleoptile h–1. The latter value is a minimum estimate for the required amount of sugar transported in the phloem of the intact seedlings, unless ethanolic fermentation was slower in the coleoptiles of intact seedlings than in the excised coleoptiles.
The operational solute translocation in anoxic rice coleoptiles contrasts with an almost complete inhibition of 2-D-deoxyglucose translocation from the scutellum to the 10-mm tip in anoxically shocked 4-d-old maize roots (Saglio, 1985). Saglio (1985) concluded the inhibition occurred during unloading in the maize root tip, rather than during transport in the phloem, hence the longer length of the translocation pathway in maize roots than in rice seedlings was presumably not responsible for the different responses by the two species. Another possible explanation is the use of anoxically shocked roots by Saglio (1985). However, there is circumstantial evidence that the limitation of sugar translocation also occurred in 2-d-old maize seedlings (estimated from data in Van Toai et al., 1995). These 2-d-old seedlings are much more tolerant to anoxic shock than are seedlings 3 d and older (Van Toai et al., 1995). Thus, phloem transport seems more intolerant to anoxia in maize roots, than in rice coleoptiles. This difference may be a specific effect on phloem translocation, but it more likely reflects widespread adverse effects on metabolism in maize roots, at least in the experiments by Saglio (1985) and Van Toai et al. (1995).
Suitability of excised coleoptiles to evaluate responses to anoxia
The present data indicate that uptake of monovalent cations (K+ and Rb+) from the external solution was inhibited to a similar degree in coleoptiles of intact rice seedlings or when excised, ‘aged’, and supplied with exogenous glucose (Table 2; for excised coleoptiles see also Colmer et al., 2001). Moreover, both systems survived anoxia for several days (data not shown), and tissue sugar concentrations were similar (83 µmol g–1 f. wt in the coleoptiles of the intact seedlings and 60 µmol g–1 f. wt in the excised coleoptiles). Growth rates in anoxia, however, were 10-fold slower in excised than in intact coleoptiles. Thus, the presently available excised system is unsuitable to evaluate how anoxia affects the rate of various growth processes. It may be possible to develop an excised system of rice coleoptiles in which the rate of growth can be experimentally manipulated. Ethylene stimulated elongation of 7-mm excised tips of rice coleoptiles (Ishizawa and Esashi, 1983) and elongation due to ethylene also occurred when O2 was at about 0·0016 mol m–3, but this latter condition has only been tested for ‘rice explants’ (seedlings from which roots had been removed; Ishizawa and Esashi, 1984).
At present, several responses to anoxia can be studied in the available excised system, which allows interpretation of processes such as ion uptake, solute leakage, and ethanolic fermentation in the coleoptile. We have developed recently an improved system for this purpose, by excising the 9-mm (approx.) tips of the coleoptiles, thus avoiding the enclosed leaf tissues and resulting in a more uniform tissue.
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ACKNOWLEDGEMENTS |
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We acknowledge Jane Gibbs, for stimulating discussions during the work, and Brian Atwell and Tim Setter, for comments on this manuscript. An anonymous referee gave useful suggestions to improve the manuscript. Shaobai Huang is grateful to UWA for a University Postgraduate Award and International Postgraduate Fee Waiver Scholarship.
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