The beneficial effect of reduced elongation growth on submergence tolerance of rice

Transcription

1 Journal of Experimental Botany, Vol. 47, No. 33, pp , October 996 Journal of Experimental Botany The beneficial effect of reduced elongation growth on submergence tolerance of rice Timothy Louis Setter ' 3 and Eufrocino Vidonia Laureles 2 Agriculture Western Australia, 3 Baron-Hay Court, South Perth, Western Australia, Australia 2 International Rice Research Institute (IRRI), PO Box 933, Manila, The Philippines Received 2 February 996; Accepted 28 May 996 Abstract Adverse effects of elongation growth on tolerance to complete submergence for up to 4 d were evaluated in rice seedlings of cultivars which differed in submergence tolerance. There is a good negative correlation between per cent survival and elongation growth of genotypes during complete submergence (r=.8). When elongation growth underwater is minimized by application of a gibberellin biosynthesis inhibitor, per cent survival increases by as much as 5 times for one cultivar. These effects are likely related to elongation growth since (i) addition of gibberellin had the opposite effect by reducing survival, and (ii) when the elongation inhibitor and gibberellin were added together, there was no effect on elongation growth and the per cent survival did not change. A GA-deficient mutant of rice which had little elongation ability during submergence showed a high level of submergence tolerance when plants were submerged at equal initial dry weights and carbohydrate levels relative to a submergence-tolerant cultivar. These results are consistent with the hypothesis that elongation growth competes with maintenance processes for energy and hence reduces survival during submergence. The impact of these findings is that in environments where elongation ability is not required, there is a potential to increase submergence tolerance of agriculturally important cultivars by selecting for least elongation, at least during periods of complete submergence. Furthermore, this trade-off between stimulated elongation growth and submergence tolerance will have important ecological consequences for the distribution of plant species in different flood-prone environments. Key words: Gibberellin, growth, Oryza sativa, rice, submergence. Introduction The flood-prone environments are planted to rice types that are adapted to excessive flooding, and rice is often the only crop able to survive in these conditions. Of these different environments, the two most important are the stagnant deepwater ecosystem (HilleRisLambers and Seshu, 982; Catling, 992) and the flash-flood affected areas of the rainfed lowland rice ecosystem (Maurya et al., 988). The mechanisms of plant adaptation to excessive flooding depend on the water regime. Where water depth rises slowly (<3cm d~') and remains at a depth of 5-4 cm for 2-4 months of the year, deepwater and floating rice cultivars are grown. These cultivars are able to elongate with floodwater and maintain shoots above water level, i.e. the mechanism of adaptation to these locations is escape from submergence via stem elongation. There are about million ha of deepwater and floating rice (IRRI, 993) and an additional 23 million ha of flash-flood prone lowland rice (calculated from Maurya et al., 988) grown at water depths up to 5 cm, relative to the world total area sown to rice of 48 million ha (IRRI, 993). In areas where rainfed lowland rice is subjected to flash-floods, elongation growth results in lodging and death of plants after the water recedes. Hence plants adapted to these areas must have submergence tolerance. There are numerous environmental factors which may limit survival of rice during submergence (Setter et al., 995). However, a dominant factor in all locations is limited gas diffusion since gases diffuse 4 times more slowly in water than in air (Armstrong, 979). The complexity of interactions to which submerged plants are exposed in relation to low CO 2 concentrations, low O 2 concentrations and high ethylene concentrations are discussed by Setter et al. (989). However, the cause of 3 To whom correspondence should be addressed. Fax: tsetter@agric.wa.gov.au 6 Oxford University Press 996

2 552 Setter and Laureles submergence tolerance, which only occurs in a few of the lowland types and not the elongating deepwater rice, remains a mystery. The impact is that after two decades of rice breeding for flood-prone areas, there has been no significant introduction of improved cultivars in these ecosystems (Maurya et al., 988). In recent research on the importance of alcoholic fermentation as an energy supply for rice coleoptiles during submergence, this metabolism was discussed in relation to its use for either (i) maintenance processes associated with survival or (ii) maintenance processes plus growth (Setter et al., 994, Green way and Setter, 996). Hence it is postulated here that by manipulating elongation growth during submergence, survival under flash-flood conditions will be altered. In deepwater rice, elongation growth of internodes during submergence is mediated by the ratio of gibberellin and ABA (Hoffmann-Benning and Kende, 992) and enhancement of gibberellin activity by ethylene (Raskin and Kende, 984; Suge, 985). Similarly, submergence induces ethylene- (Voesenek et al., 993) and gibberellinmediated (Blom et al., 994; Voesenek et al., 996) elongation growth for survival during submergence of wetland species of the dicotyledonous plant Rumex. Application of exogenous gibberellin or gibberellin biosynthesis inhibitors was therefore expected to affect some of the same physiological processes which are normally involved in elongation of plants exposed to submergence. Work presented here focuses on how survival of established rice seedlings is affected by the successful manipulation of elongation growth during submergence. This is achieved using the growth regulator, gibberellin (GA 3 ), to increase growth and a gibberellin biosynthesis inhibitor (paclobutrazol) to reduce growth during submergence. Comparisons are also made with the response of a GA-deficient mutant to submergence. Materials and methods Plant material Six cultivars ofrice [Oryza sativa (L.)] were used. Three lowland rice cultivars which differed in submergence tolerance were used: FR3A, IR42 and Calrose (Mazaredo and Vergara, 982; Setter et al., 994); and two deepwater rice cultivars with good elongation ability: HTA6 (Sittiyos et al., 988) and PG56 (Kupkanchanakul et al., 988). The GA-deficient mutant, dwarf, lowland cultivar Tan-ginbozu (Suge and Murakami, 968) was used in some experiments. A larger population of 93 cultivars was also evaluated from the RRI Genebank database for all cultivars which had ever been screened for submergence tolerance and elongation ability. Submergence tolerance and elongation capacity were scored with a scale ranging from to 9. A submergence score of, 3, 5, 7, and 9 represents % and above, 95-99, 75-94, 5-74, and -49% survival during submergence, respectively, based on a comparative scale relative to a tolerant check cultivar like FR3A (IRRI, 988). An elongation score of represents elongation equal to an elongating floating rice cultivar (a cultivar capable of growing at > m water depth) during flooding, 5 is elongation equal to an elongating semi-dwarf cultivar like IR4 (Mazaredo et al., 996), while 9 represents little or no elongation (IRRI, 988). Experimental procedures Seeds were germinated for h in aerated water in the dark at 3 C. Germinated seeds were sown cm deep in trays 4 x 3 x 2 cm deep containing approximately 6 kg Maahas clay soil (Ponnamperuma, 984). Soil was flooded to approximately cm depth at week after sowing. Seedlings were grown in the glasshouse, and 4-d-old seedlings were used in all experiments. At least 3 seedlings with a minimum of three replicate trays were used in all experiments. Trays were fitted with wire mesh racks with 3x3 cm mesh approximately 5 cm above the surface which prevented the seedlings from becoming dislodged from the soil (lodging) when trays were removed from the submergence tank. The duration of submergence varied over several days in different experiments since the natural irradiance before and during submergence differed, and this is known to affect submergence tolerance of rice (Palada and Vergara, 972). While differences in irradiance affected the time plants could tolerate submergence, the genotypic ranking of cultivars remained the same (Palada and Vergara, 972; Setter and Laureles, unpublished data). Hence plants were usually de-submerged between 7-4 d after submergence to optimize differences between genotypes. During all submergence treatments except for Fig. 2, plants were maintained completely submerged in water with CO 2 concentrations ranging from. to.2 mol m~ 3 at ph The floodwater O 2 concentrations ranged from.6 to.8 and from.2 to.3 mol m" 3 at h and h, respectively. Plants were exposed to natural irradiance as stated in the figures with sunshine hours d~'. Plant survival was measured 7 d after de-submergence and is defined as the ability of a seedling to produce new leaves and continue to grow following treatments. Plant elongation was measured as the increase in plant height which occurred from the beginning to the end of the submergence period. For the large submergence screening shown in Fig. 2, there were 93 cultivars or cross-breds grown in Maahas clay (as above) for 4 d, and seedlings were completely submerged in glasshouse tanks or ponds in the field. Data in Fig. 2 are the combination of numerous experiments at different times of the year, and there is no information on irradiance or floodwater environmental conditions during submergence. The gibberellin biosynthesis inhibitor, paclobutrazol (Rademacher, 992), was routinely applied to either floodwater at -5 mg I" or as a spray at 2 mg I" ; for sprays approximately ml of solution was sprayed per plant 24 h prior to submergence. In preliminary experiments, the commercial paclobutrazol solution applied to floodwater at 5,, and 5 mg r' gave similar effects on elongation, however, at concentrations of mg I" and above thefloodwaterbecame turbid, anoxic and all plants died within 2 4 d (data not presented). Hence only data for floodwater at -5mg ~" or for leaf sprays at 2 mg P paclobutrazol are reported here. Commercial paclobutrazol solution (Cultar 25 SC, 25% (w/v) paclobutrazol, 99.99% pure) was used in all experiments and was obtained from Zeneca Agrochemicals (Jardine Agchem, Philadelphia). Pure paclobutrazol (courtesy of Dr JR Lenton, AFRC, University of Bristol, Long Ashton, UK) was used in two experiments and confirmed that the effects of the commercial

3 solution were quantitatively reproducible in relation to shoot length elongation and survival (data not presented). In some experiments gibberellin (GA 3, Sigma Chemical Co, MO, USA) was applied to leaves as a spray at 5-2 mgt as above. In other experiments where GA 3 and paclobutrazol were added together, plants were first sprayed with paclobutrazol, allowed to dry and then sprayed with GA 3 as above. Metabolic measurements Effects of GA 3 and paclobutrazol on single leaf photosynthesis rates of intact plants were measured with a Li-Cor portable photosynthesis system (LI-62, Li-Cor, Nebraska, USA) using a 2 cm 3 leaf chamber and the uppermost fully expanded leaf at saturating irradiance. Plants were harvested and assayed for carbohydrates using samples which were fresh frozen in liquid N 2 and then freeze-dried (Setter et al., 994). Samples were assayed for soluble sugars using anthrone reagent, and starch by hydrolysis with amyloglucosidase, followed by glucose assay using glucose oxidase (Sigma Chem Co., MO, USA). Results Genotypic variation in elongation and survival Survival of five rice cultivars was negatively correlated with elongation growth during complete submergence, with deepwater rice cultivars tending to have the lowest survival and greatest elongation relative to lowland cultivars (Fig. ). These results were not a consequence of differences in initial shoot length since shoot lengths (±sem) of FR3A, IR42, Calrose, HTA6 and PG56 at time of submergence were 39.3 ±2.3, 33. ±.5, 37. ± ±.2, and 35.8 ±2.4 cm, respectively. This negative relationship between submergence tolerance and elongation during submergence was confirmed in a larger population of 93 cultivars or cross-breds taken from the IRRI Genebank database (Fig. 2). Of 23 cultivars or cross-breds which had a submergence toleriwu - a ' D i N sl \ O y = -O.376x + - «TX D O A T FR3A IR42 Shoot Length (% of original) Calross HTA6 PG56 Fig.. Shoot elongation of rice cultivars during submergence (±sem) relative to survival (% ±sem). Shoot length is expressed as a % of original shoot length at submergence. Plants were submerged 4 d at a mean daily irradiance of 22.3 MJ m" 2. Open symbols, lowland rice cultivars; closed symbols, deepwater rice cultivars. SHORT + 9 Is CD "5 83 LONG 2 2 Submergence tolerance of rice GOOD Decreasing submergence tolerance POOR Fig. 2. Scoring of all germplasm accessions which have been tested for submergence and for elongation (93 cultivars or cross-breds). Data obtained from IRRI Genebank database. The numbers within the axes refer to the number of cultivars or cross-breds with a given combination of submergence tolerance and elongation character. ance score of (% survival relative to the submergence-tolerant check FR3A, IRRI 988) elongation score was only 5-7 (poor); while all 69 cultivars or crossbreds which had a good elongation score of (elongation equal to a tolerant check; IRRI, 988) had a poor submergence tolerance score of 9 (-49% survival relative to tolerant check FR3A). It may be important that 49% of the entire population screened were in the slowest two elongating categories (7 and 9) and in the least tolerant submergence categories. However, there were no cultivars or cross-breds which had good elongation plus good submergence tolerance (Scores -3, Fig. 2). A chi-square analysis of these data predicts that the random chance that all 69 cultivars or cross-breds with good elongation (Score=l) in Fig. 2 will have the worst submergence tolerance score of 9 has a probability of only.2. Manipulation of elongation using growth regulators Calrose and HTA6 had the same shoot length increase of about 22% during submergence (Fig. ), and these two cultivars were selected for further analyses. When plants were submerged in 5 mg ~' paclobutrazol or sprayed with 2 mg I" paclobutrazol prior to submergence they had lower elongation of shoots and survival increased by 74-98% relative to plants without paclobutrazol treatment (Fig. 3). When elongation and survival were plotted during 7 or d submergence and plants were submerged in floodwater containing, 5, or 5 mg I" paclobutrazol, a high correlation occurred between submergence tolerance and shoot elongation (Fig. 4), as was previously observed for cultivars that

4 554 Setter and Laureles CalroM Cultivar HTA6 (27) OmgL - tmgl " PB8PRAY Fig. 3. Survival (±sem) of cultivars during d submergence. Paclobutrazol (PB) treatments (dark bars) were used to reduce elongation growth; spray was at 2 mg I". Figures in parentheses are shoot length as % of length before submergence; shoot lengths at time of submergence were 32. ±. cm for both Calrose and HTA6. The sems for PB spray treatments were both ±% A fu (All 5) v ) A A y= -2 7A+369 r A(AII) Calrose HTABO Shoot length (% of original) Fig. 4. Shoot elongation manipulated by paclobutrazol treatments during submergence relative to survival of rice cultivars during submergence. Shoot length is expressed as a % of original shoot length at submergence; shoot lengths at time of submergence are as in Fig. 3. Plants were submerged 7- d at a mean daily irradiance of 4.9 MJ m~ 2. Symbols as per Fig. ; figures in parentheses aremg I" paclobutrazol in floodwatcr. naturally differed in elongation during submergence (cf. Figs and 4). Effects of elongation were more pronounced and the slope became more negative under conditions where there was low light. For example, in Fig. the mean irradiance over -3 to + d after submergence was 22.3 ±5.5 MJm" 2 under natural conditions, and -2% survival occurred when shoot length increased to 25% of initial values. In contrast, during conditions which prevailed in Fig. 4, the mean irradiance was 4.9 ±5.4, and -2% survival occurred when shoot length increased to only 5% of initial values. The five cultivars shown in Fig. had greater survival PS PB during submergence in a total of six experiments, when plants were sprayed with paclobutrazol at 2 mg I", or submerged at 5-5 mg ~ paclobutrazol, than for plants without paclobutrazol, though significant differences usually only occurred when survival of non-treated plants was <65% (data not presented). In one experiment paclobutrazol increased the time for 5% survival during submergence from 7 d to 2 d for the floating rice cultivar PG56 (data not presented), and this was equivalent to the time for 5% survival of the submergence-tolerant cultivar FR3A without paclobutrazol treatment (cf. Fig. without paclobutrazol treatment). One cultivar with the lowest elongation during submergence and the greatest submergence tolerance, FR3A (Fig. ), was selected to evaluate the effects of decreasing or increasing elongation during submergence. Plants treated with paclobutrazol had reduced elongation growth and increased percentage of survival to ±% (Fig. 5); however, the effects of paclobutrazol on increasing survival were not as pronounced relative to other cultivars where there were large reductions in survival of nontreated plants during submergence (cf. Fig. 5 and Fig. 3). In contrast, FR3A plants treated with gibberellin had about double the shoot elongation of non-treated (control) plants during submergence, and this resulted in a decrease in survival from 85% to 35% (Fig. 5). The beneficial effects of paclobutrazol on submergence tolerance were probably the result of reducing elongation growth rather than other effects, e.g. on photosynthesis, which might be associated with this growth regulator (see Discussion). This was demonstrated by applying paclobutrazol spray at 2 mg I' to plants which were also sprayed with, 5, 5, or 2 mg I" gibberellin. With increasing concentrations of gibberellin, there was greater n - (6) (2) (35) I Treatment El Control +PB +GA Fig. 5. Effects of gibberellin (5 mg I"') or paclobutrazol (2 mg I"') sprays on % survival (±sem) of the submergence tolerant rice cultivar FR3A. Plants were sprayed 24 h before submergence and submerged 4 d. Shoot length after submergence (% of initial length prior to submergence) is given in parentheses; shoot length at time of submergence was 37. ±3.3cm. The sem for PB spray treatment was ±%.

5 elongation during submergence and greater reduction in survival, even though all plants were treated at the same concentration of paclobutrazol (Fig. 6). Plants sprayed with paclobutrazol at 2 mg ~ l plus gibberellin at 5- mg I" had the same elongation growth as that of non-sprayed plants (closed and open symbols, respectively, Fig. 6). They also had the same per cent survival during submergence; this was demonstrated for both Calrose and HTA6 (Fig. 6A and B, respectively). There were also no significant effects of spraying plants with 2 mg ~ l gibberellin or paclobutrazol on net single leaf photosynthesis for non-submerged plants for either Calrose or HTA6 over 6 42 h after spraying treatments (Table ). Survival during complete submergence was also measured in a GA-deficient mutant Tan-ginbozu relative to a submergence-tolerant (FR3A) and -intolerant check Submergence tolerance of rice 555 Table. Effects of spraying leaves with 2 mg I' gibberellin or paclobutrazol on single leaf photosynthesis (Pn) Data are the means of measurements at 6, 2 and 42 h after treatments. Treatment Non-sprayed (control) Paclobutrazol Gibberellin LSD (P<.5) Single leaf Pn (firnol CO 2 m~ 2 Calrose HTA (IR42). In this experiment, plants of FR3A and IR42 were grown for or 4 d before submergence so as to span the seedling dry weight of the dwarf cultivar Tanginbozu (Table 2). The GA-deficient mutant had intermediate survival when 4-d-old seedlings were compared with 4-d-old submergence-tolerant and -intolerant check cultivars. This was largely the result of the low initial seedling dry weight of the dwarf Tan-ginbozu seedlings; all seedlings had the same per cent total carbohydrates (±sem; soluble sugars plus starch) of on a dry weight basis. When submergence tolerance was evaluated using seedlings with the same dry weight as the submergence-tolerant and -intolerant checks (cf. 4 d Tanginbozu and IR42 with d FR3A), Tan-ginbozu had an intermediate level of elongation and a high percentage of survival equal to FR3A (Table 2). Furthermore, there was no significant effect of paclobutrazol on elongation growth or per cent survival of Tan-ginbozu during submergence (data not presented). 8 - E IE Shoot length Increa&a (cm) <W) Discussion Greater leaf extension during submergence was originally associated with an intolerant rice cultivar IR42, relative to reduced leaf extension which occurred in two tolerant cultivars, FR3A and Kurkaruppan, during submergence (Jackson et ai, 987). These results were thought to be Table 2. Growth, elongation during submergence and submergence tolerance (±sem) of a GA-deficient rice cultivar, Tan-ginbozu, relative to submergence tolerance checks Plants were grown for or 4 d and then submerged for d Shoot length Increase (cm) Fig. 6. Survival (%±sem) of Calrose (A) and HTA6 (B) when shoot length increase of plants is manipulated during submergence. All plants were submerged for 4 d. Plants were either not sprayed (open circles) or sprayed with elongation inhibitor paclobutrazol at 2 mg I" (PB, closed symbols) before submergence. PB-treated plants were also sprayed with or without an elongation promoter gibberellin (GA) at mg I" indicated in parentheses. Shoot lengths at time of submergence were 29.4 ±2.2 and 25.7 ±.8 cm for Calrose and HTA6, respectively. Cultivar (seedling age) Dry weight (mg seedling" ) Tan-ginbozu (GA-deficient 4 d 6±7 FR3A (tolerant check) d 9±4 4 d 29 ±42 IR42 (intolerant check) d 66± 4 d 26±27 Elongation (cm; % original length) mutant) 9.7±2.( 32) 9.5±3.O(33) 3.5±.3(43) 2.7±.() 27.2±2.7(3) Survival (%) 72±2 74±3 93±9 8± 4±3

6 556 Setter and Laureles due to differences in responsiveness to ethylene, and they were associated with greater chlorosis of older leaves during submergence and a likely increased susceptibility to lodging of the intolerant cultivar when water levels recede. In other work, the importance of slow growth during flooding was suggested as a means to maintain high carbohydrate supply and hence prolonged energy supply in rice (Setter et al., 987, 994). This response was subsequently reviewed by Greenway and Setter (996) as one of several mechanisms of tolerance to submergence and waterlogging of plants. Slow growth during submergence may be beneficial since energy and carbohydrates can be used for either (i) maintenance processes essential for survival including protein turnover and active transport processes to maintain ion concentrations (Penning de Vries, 975), or (ii) growth plus maintenance processes. The response of seeds of 2 crop species to low O 2 supply was similarly grouped according to whether species stopped germination and maintained a low adenylate energy charge for prolonged energy supply or they continued germination with a high energy charge (Al-Ani et al., 985). Under anoxia, some plants with small seeds and a low carbohydrate supply have low metabolic rates and hence long-term survival; this applies to imbibed lettuce seeds which were reported to survive up to 3 weeks in anoxia (Raymond and Pradet, 98) and for Echinochloa crus-pavonis seeds which survived up to 3 d under anoxia (Zhang et al., 994). The beneficial effects of reduced elongation growth of plants during submergence was demonstrated here by four observations: (i) comparison of submergence tolerance in cultivars which naturally varied in elongation during submergence (Figs, 2), (ii) beneficial effects of reducing elongation growth using the gibberellin biosynthesis inhibitor paclobutrazol (Figs 3, 5), (iii) adverse effects of increasing elongation growth using exogenous gibberellin (GA 3, Fig. 5), and (iv) observations of a high level of submergence tolerance in a GA-deficient mutant which had low absolute increases in elongation during submergence (Table ). Results presented here for established seedlings may differ at other stages of development. For example, elongation of rice coleoptiles is not inhibited by inhibitors of GA biosynthesis (Hoffmann-Benning and Kende, 992). However, internodal elongation growth of deepwater rice was reduced by tetcyclacis (Raskin and Kende, 984), which inhibits the GA biosynthesis pathway at the same site of kaurene oxidase as paclobutrazol (Rademacher, 992) used here. It is likely that the beneficial effects of paclobutrazol occurred via inhibition of kaurene oxidase in the gibberellin biosynthesis pathway (Rademacher, 992). However, it is possible that the up to 5-fold increase in survival of submerged plants treated with paclobutrazol (Fig. 3) was due to other effects of this growth regulator aside from reductions in elongation growth. For example, paclobutrazol was previously shown to increase leaf photosynthesis by 22% in sugar beet (leaf area basis, Dalziel and Lawrence, 984). Such effects were not demonstrated here (Table ). The most convincing evidence that paclobutrazol acted via changes in elongation growth is that when plants were treated with paclobutrazol plus increasing concentrations of gibberellin, the elongation growth was re-established and survival was proportionally reduced (Fig. 6). Furthermore, when paclobutrazol plus gibberellin-treated plants had the same elongation as that of non-treated plants, they also had the same per cent survival during submergence (for 2 cultivars, Fig. 6A, B). Thefinding that the submergence tolerance of the GA-deficient mutant Tan-ginbozu is equivalent to that of FR3A when seedlings of equal initial dry weight (Table 2) and equal carbohydrate levels (Results) are compared, also supports the view that slow elongation growth during submergence is beneficial to survival. Out of 8,5 cultivars screened in the IRRI Genebank database only 2.% have submergence tolerance equivalent to that of FR3A (cf. Fig. 2), hence the probability that Tan-ginbozu was submergence tolerant by chance is unlikely. Other experiments demonstrated that for Tan-ginbozu, paclobutrazol had no effect on elongation during submergence, nor did it significantly increase submergence tolerance of Tan-ginbozu. The mechanism for how submergence affects shoot elongation and possible emergence via ethylene accumulation which stimulates GA biosynthesis or increases sensitivity to GA in rice and Rumex is well described (Introduction). However, it is unclear why changes in GA biosynthesis affect survival during continuous complete submergence. Presumably reduced elongation growth has an effect of enabling greater energy supply at any one time or prolonging supply of reserves of carbohydrates available for maintenance processes. This is consistent with the response of FR3A, which has the lowest elongation during submergence (Fig ), and hence the least effects of paclobutrazol on increasing survival (Fig. 4). This hypothesis of a greater energy supply with reduced growth would be supported by comparing timecourses in carbohydrate concentations of plants with and without elongation inhibitors during submergence. Alternatively carbohydrates may be the same in both treatments, but the synthesis of anaerobic proteins may occur at a faster rate where there is less demand on energy supply for growth. Considerable information is available on the relationship between energy requirements for growth and for maintenance processes in non-submerged plants. In nonsubmerged rice, the energy requirements are indicated by respiration which is the sum of maintenance respiration and growth respiration. Maintenance respiration in rice varies depending on the stage of development, but it generally accounts for 22-33% of the respiration in

7 Submergence tolerance of rice 557 rapidly growing seedlings and tillering plants, and up to 95% in plants at maturity (Yamaguchi, 978; Penning de Vries et al., 989). Such values would suggest that up to 3-fold increases in survival might occur by complete cessation of growth of plants before flowering. This estimate is a maximum value since (i) it assumes that maintenance processes remain the same during submergence and (ii) some elongation growth occurs for even the most tolerant cultivars like FR3A (Figs,5). The former is unlikely since there is usually a positive relationship between maintenance respiration and plant relative growth rate (Penning de Vries et al., 983); and even for non-growing beetroot tissue exposed to anoxia for more than d, the maintenance requirements are reduced by to 25 times relative to aerated tissues (Zhang and Greenway, 994). Additional 'maintenance' during submergence would include the energy requirements associated with synthesis of anaerobic proteins and maintenance of membrane integrity. For example, the synthesis of anaerobic proteins is an important adaptive feature of rice (Ricard and Pradet, 989) and maize (Bailey-Serres et al., 988) exposed to anoxia. That some elongation growth occurs for FR3A implies that submergence tolerance of even this cultivar could be improved by growth reductions during submergence; this was supported by the beneficial effects of paclobutrazol on this cultivar in two experiments. It remains possible that GA could be a negative regulator of a submergence stress-inducible gene. This would be consistent with the effects of paclobutrazol on submergence tolerance and the response of the GA-deficient mutant. Extensive research has demonstrated that ABA antagonizes the GA-induced transcription of a-amylase genes in cereal aleurone layers (reviewed by Chandler and Robertson, 994), and similar effects could occur for GA in rice during submergence. The mechanism for beneficial effects of reduced GA metabolism in rice during submergence requires further research, however, advantage can already be taken of the impact for enhancing agricultural production. The opportunity for increasing submergence tolerance of rice by selecting for reduced shoot elongation during submergence offers an easy selection criterion for plant breeding programmes. Recent work on the physiology and genetics of submergence tolerance of rice highlights several opportunities for molecular approaches to increasing submergence tolerance based on physiological results presented here (Setter et al., 996). Increasing submergence tolerance by reduction in growth demonstrates the importance of optimizing metabolism related to adaptation, maintenance and growth processes in plants exposed to adverse environments. Results presented here also have ecological significance. In flood-prone areas, there will likely be a strong selection pressure for native species which have greater survival by either submergence tolerance or elongation ability during short- or long-term flooding, respectively. Hence, the optimum mode of survival will depend on the water regimes of the specific habitat. Two modes of adjustment to flooding occur in the genus Rumex by life-cycle timing and phenotypic plasticity including shoot elongation ability (Blom et al., 994; Voesenek and Blom, 996). In two species from frequently flooded areas which were exposed to partial or complete submergence, survival of R maritimus was restricted mainly to plants which emerged from the water by shoot elongation. In comparison, R. palustris had a greater submergence tolerance and it also had an ability to elongate, though in several cases this was less than for R maritimus (van der Sman et al., 993). It would be useful to clarify whether these differences in response between species are due to differences in development, including carbohydrate status prior to flooding, or are partly a consequence of the trade-off between elongation growth and submergence tolerance as shown here for rice. The combination of both elongation ability and submergence tolerance in one genotype is possible under certain conditions. For example, it would be of adaptive advantage for plants to have a metabolic switch optimizing the response to flooding by (i) enhanced elongation ability when floodwater increases slowly resulting in partial submergence, as it often does in deepwater and floating rice ecosystems (Catling, 992), and (ii) reduced elongation ability with consequently greater submergence tolerance when floodwater increases rapidly resulting in complete submergence, as often occurs during flashflooding. Whether this fine control of elongation ability and submergence tolerance already exists in some aquatic and semi-aquatic plants like rice is unknown. Acknowledgements We thank Associate Professor Hank Greenway for stimulating discussions during the experimental work and reviewing an early draft of the manuscript; Drs Peter Chandler and Marc Ellis for helping to conceptualize interpretations of involvement of gibberellin metabolism; Ms G Loresto and M Oliva for assistance with Genebank database information; Dr H Suge (Tohoku University, Japan) and Dr S Akita (University of Tokyo, Japan) for seed of the dwarf-mutant Tan-ginbozu; Dr LACJ Voesenek for information on Rumex species; Ms A Mazaredo, Mr R Parao, M del Valle, D Reano, and P Victoria for glasshouse assistance. References Al-Ani A, Bruzau F, Raymond P, Saint-Ges V, Leblanc JM, Pradet A Germination, respiration and adenylate energy charge of seeds at various oxygen partial pressures. Plant Physiology 79, Armstrong W Aeration in higher plants. In: Woolhouse

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