Skip to main content
NCBI home page
Annals of Botany logoLink to Annals of Botany
. 2014 Jan 14;113(5):851–859. doi: 10.1093/aob/mct305

Rice alcohol dehydrogenase 1 promotes survival and has a major impact on carbohydrate metabolism in the embryo and endosperm when seeds are germinated in partially oxygenated water

Hirokazu Takahashi 1,*, Hank Greenway 2, Hideo Matsumura 3, Nobuhiro Tsutsumi 4, Mikio Nakazono 1
PMCID: PMC3962239  PMID: 24431339

Abstract

Background and Aims

Rice (Oryza sativa) has the rare ability to germinate and elongate a coleoptile under oxygen-deficient conditions, which include both hypoxia and anoxia. It has previously been shown that ALCOHOL DEHYDROGENASE 1 (ADH1) is required for cell division and cell elongation in the coleoptile of submerged rice seedlings by means of studies using a rice ADH1-deficient mutant, reduced adh activity (rad). The aim of this study was to understand how low ADH1 in rice affects carbohydrate metabolism in the embryo and endosperm, and lactate and alanine synthesis in the embryo during germination and subsequent coleoptile growth in submerged seedlings.

Methods

Wild-type and rad mutant rice seeds were germinated and grown under complete submergence. At 1, 3, 5 and 7 d after imbibition, the embryo and endosperm were separated and several of their metabolites were measured and compared.

Key results

In the rad embryo, the rate of ethanol fermentation was halved, while lactate and alanine concentrations were 2·4- and 5·7- fold higher in the mutant than in the wild type. Glucose and fructose concentrations in the embryos increased with time in the wild type, but not in the rad mutant. The rad mutant endosperm had lower amounts of the α-amylases RAMY1A and RAMY3D, resulting in less starch degradation and lower glucose concentrations.

Conclusions

These results suggest that ADH1 is essential for sugar metabolism via glycolysis to ethanol fermentation in both the embryo and endosperm. In the endosperm, energy is presumably needed for synthesis of the amylases and for sucrose synthesis in the endosperm, as well as for sugar transport to the embryo.

Keywords: alcohol dehydrogenase, ADH1, submergence tolerance, carbohydrate metabolism, embryo, endosperm, germination, reduced ADH activity, rice, Oryza sativa

INTRODUCTION

Seeds of rice, unlike those of other gramineous crops, are able to germinate by means of coleoptile elongation under both hypoxia (Atwell et al., 1982; Alpi and Beevers, 1983) and anoxia (Atwell et al., 1982; Alpi and Beevers, 1983; Perata et al., 1997; Magneschi and Perata, 2009). Under oxygen-limiting conditions, plants must rely on glycolysis for ATP production (Perata and Alpi, 1993; Gibbs and Greenway, 2003; Bailey-Serres and Voesenek, 2008). Thus, glycolysis-mediated ATP production plays an important role in germination of rice seeds under hypoxia–anaerobiosis.

Oxygen deprivation is associated with increased ethanol fermentation [catalysed by pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH)] and lactate fermentation [catalysed by lactate dehydrogenase (LDH)] and increased biosynthesis of alanine, γ-aminobutyric acid (GABA), succinate and occasionally malate (Davies, 1980; ap Rees et al., 1987; Gibbs and Greenway, 2003; Bailey-Serres and Voesenek, 2008). In rice coleoptiles subjected to anoxia, glycolysis is mainly linked to ethanol fermentation, with only a small percentage of the carbon flowing to alanine and lactate (Kato-Noguchi, 2006). Sugar levels of the coleoptile are similar in anoxic and aerated solutions (Atwell and Greenway, 1987). Exogenous sugar does not appreciably stimulate coleoptile elongation of intact rice seedlings (Atwell and Greenway, 1987), including those of two anoxia-tolerant cultivars (Huang et al., 2003). Further, in anaerobic endosperm, the activities of starch-degrading enzymes (α-amylase, α-glucosidase, and debranching enzyme) are induced in rice seeds, but not in wheat and barley seeds (Perata et al., 1992, 1993, 1997; Guglielminetti et al., 1995). The supply of external glucose allows wheat seeds to begin germination under anaerobic conditions, but there is no further development (Perata et al., 1992). Together, these results suggest that adequate ATP formation in the endosperm is important for amylase synthesis, as well as for sucrose synthesis and its translocation from the endosperm to the embryo.

In the early stage of rice seedlings, the overexpression of ADH or PDC did not improve root growth under oxygen-deficient conditions (Agarwal et al., 2007). However, it was reported that ADH is involved in coleoptile growth under oxygen-limiting conditions, and rice coleoptile growth is depressed by deficiencies of ADH (Matsumura et al., 1995, 1998; Rahman et al., 2001; Edwards et al., 2012). The rice reduced adh activity (rad) mutant possesses a point mutation in the ADH1 gene and has low ADH activities (Matsumura et al., 1995, 1998; Saika et al., 2006). In a genotyping analysis of the F2 population from a cross between the rad mutant and the rice cultivar Kasalath, the point mutation of the rice ADH1 gene was found to be associated with a decrease in coleoptile length under submergence (Saika et al., 2006). Importantly, under aerobic conditions, coleoptile elongation was similar in the mutant and wild type (Takahashi et al., 2011). The rice genome has three ADH genes (ADH1, ADH2 and ADH3; Xie and Wu, 1989; Lasanthi-Kudahettige et al., 2007; Terada et al., 2007). Interestingly, the mutation of ADH1 prevented coleoptile elongation during submergence, but coleoptile elongation in the ADH2 mutant was not suppressed under submerged conditions (Terada et al., 2007). In the rad mutant, the rate of ethanol formation by the coleoptile was reduced under anoxia (Edwards et al., 2012). Interestingly, these rad mutant phenotypes were observed not only under anaerobic (anoxic) conditions, but also under hypoxic conditions, such as those in a semi-stagnant solution, even when it contains some oxygen (Saika et al., 2006; Takahashi et al., 2011). In the present experiments, oxygen deficiency was usually imposed by sowing seeds in semi-stagnant solutions, i.e. without forced turbulence. These semi-stagnant solutions simulate the O2 regime in many field situations and lead to anoxic zones with anaerobic metabolism (reviewed by Gibbs and Greenway, 2003). These O2 deficits have also been found for a range of seeds germinating in stagnant conditions (Al-Ani et al., 1985; Raymond et al., 1985).

The present investigation focuses on the question of whether the low ADH in the mutant affects sugar, lactate and alanine concentrations in the embryos (which include the emerging coleoptile). We also investigated whether the low ADH in the mutant decreases the synthesis of amylases and sucrose concentration in the endosperm, which are important to sugar translocation to the coleoptile.

MATERIALS AND METHODS

Plant materials and growth conditions

The rice (Oryza sativa) rad mutant and its wild type (‘Kinmaze’) were used in this study. Dehulled caryopses (seeds) of rice were sterilized in a 0·6 % (v/v) sodium hypochlorite solution for 30 min. After washing with deionized water five times, 15 seeds (ten seeds in the case of seeds used for ethanol measurement) were placed on the bottoms of 1 L glass bottles filled with 1 L of semi-stagnant deionized water. Semi-stagnant water is defined as water that is not mechanically mixed but still has some mixing due to convection currents. This distinguishes the water from the stagnant water used by Wiengweera et al. (1997), in which all convection was suppressed by the addition of 0·1 % agar to the water. In our conditions, water had an appreciable O2 concentration, but the absence of forced turbulence ensured that O2 diffusion, which is 104-fold slower in solution than in air (Armstrong, 1979), was the main means of O2 transport. The lids of the bottles were tightly closed to prevent the loss of ethanol and entry of O2. The seeds were germinated and grown under complete submergence in darkness at 28 °C. The dissolved oxygen (DO) concentration of water in the glass bottles was measured with a DO electrode (SevenGo pr SG6, Mettler Toledo, Schwerzenbach, Switzerland). The electrode was carefully immersed in the water so as not to stir the water. The DO concentration was 0·216 ± 0·002 mm (mean ± s.d.), and gradually decreased to 0·132 ± 0·008 mm in the wild type and to 0·200 ± 0·007 mm in the rad mutant at 7 d after germination. For the time-course experiments, independent glass bottles were used for each time, and seedlings were harvested. Dry seeds were used as samples at time zero (i.e. at 0 d). We separated embryo and endosperm from the dry seeds or the geminated seeds, and measured their fresh weights. In this study, embryo is defined as the emerging coleoptile, root and scutellum of a rice seedling, i.e. it includes all of the seedling except the endosperm. Subsequently, the isolated embryo and endosperm were freeze-dried (Freezone 1, Labconco Corporation, Kansas City, MO, USA) and their dry weights were measured.

A separate experiment was conducted to determine the effects of an anoxic treatment on growth and ethanol production (see Supplementary Data Methods).

For viability testing during submergence, seeds were germinated and grown under complete submergence in darkness at 28 °C for 7 or 14 d. Subsequently, the seedlings were transferred to moist filter paper in air and were grown for 7 d with light provided (photosynthetically active radiation: 200–250 µmol m−2 s−1) at 28 °C. The numbers of recovered plants were assessed by the emergence or otherwise of the leaf. Survival rates of seedlings were calculated by dividing the numbers of recovered seedlings by the total numbers of seedlings.

To determine the effect of exogenous sugar, five seeds were placed on the bottom of six 100 mL glass bottles filled with 100 mL of deionized water with sugar as follows: three bottles had 90 mm glucose and three bottles had 90 mm sucrose. Seeds were germinated and grown for 3 d.

Extraction of metabolites

Freeze-dried embryos were ground with a Multi-Beads Shocker (Yasui-Kikai Co. Ltd, Osaka, Japan) for extractions of soluble sugars, l-lactate and l-alanine, and endosperm were ground for extractions of soluble sugars and starch. After grinding, soluble sugars were extracted by boiling in 80 % (v/v) ethanol twice. The solution was centrifuged at 12 000 g for 15 min. The supernatant was completely dried by a Micro Vac MV-100 rotary evaporator (TOMY, Tokyo, Japan). After the extraction of soluble sugars from the endosperm, the pellet (i.e. debris containing starch) was solubilized by boiling in 200 mm KOH for 2 min and the solubilized starch was cooled to room temperature. Subsequently, the pH was adjusted to 6–8 by 18 % (v/v) acetic acid. l-Lactate and l-alanine were extracted by 5 % HClO4 on ice for 1 h. The supernatant was collected by centrifuging (12 000 g, 15 min, 4 °C) and neutralized to pH 6–8 by 5 n KOH.

Determination of metabolites

To measure the ethanol concentration from fermentation, ten rice seedlings (average mass: 0·38 ± 0·09 g) were submerged and grown in 1 L of semi-stagnant water for 1, 3, 5 or 7 d. Cell membranes are highly permeable to ethanol so that nearly all of the ethanol produced by fermentation escapes to the medium (Davies, 1980; Gibbs and Greenway, 2003). The ethanol concentration in the water was determined by the generation of NADH during a coupled assay of ADH and aldehyde dehydrogenase (Beutler, 1983) as modified by Colmer et al. (2001). NADH was measured at 340 nm with a spectrophotometer (DU®640, Beckman, Coulter, Fullerton, CA, USA).

l-Lactic acid, starch and soluble sugar (i.e. sucrose, d-glucose and d-fructose) concentrations were determined with TC l-Lactic Acid, TC Starch and TC Sucrose/d-Glucose/d-Fructose kits (Roche Diagnostics, Mannheim, Germany), respectively. Alanine was converted to pyruvate by adding glutamate-pyruvate transaminase and 2-oxoglutarate to the reaction mixture, and the alanine concentration was calculated by determining the concentration of the converted pyruvate using a TC Pyruvate kit (Roche Diagnostics). The sugar concentrations in a sample of seedlings were expressed as the number of millimoles divided by the volume of water (litres) in tissues, with water content calculated by subtracting dry weight from fresh weight.

Protein extraction and enzyme assay

Protein was extracted from embryo and endosperm following Matsumura et al. (1998). The total soluble protein concentration in each extract was determined with a Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Richmond, CA, USA) according to the manufacturer's protocol.

The ADH activity was assayed by determining the reduction of NAD+ to NADH during the conversion of ethanol into acetaldehyde at 25 °C in a reaction mixture containing 0·15 m Tris–HCl buffer (pH 8·0), 0·3 mm NAD+, 30 mm ethanol and 30 µL of enzyme extract, as described by Xie and Wu (1989).

Western blotting

Polyclonal antibodies against rice ADH (Kadowaki et al., 1988) were provided by Dr K. Kadowaki (National Institute of Crop Sciences, Tsukuba, Japan). Polyclonal antibodies against RAMY1A and RAMY3D were purchased from Agrisera (Vännäs, Sweden).

Total protein was denatured and separated by SDS–PAGE. Each lane was loaded with 1 µg of protein for detection of ADH and 10 µg of protein for detection of RAMY1A and RAMY3D. Acrylamide concentrations were 4·75 and 10 % (w/v) in the stacking and separation gels, respectively. Proteins were transferred to an Immobilon™-P membrane (Millipore, Bedford, MA, USA). Rice ADH-, RAMY1A- and RAMY3D-specific antibodies were used for immunodetections. Signals were detected using Lumi-light plus (Roche) and LAS-1000 (GE Healthcare UK Ltd, Buckinghamshire, UK).

RNA extraction and RT–PCR

Total RNA was extracted from the frozen endosperm using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). Transcript levels of RAMY1A, RAMY3D (α-amylase gene) and Ubq were measured by semi-quantitative reverse transcription–PCR (RT–PCR). First-strand cDNA was synthesized using Superscript III (Invitrogen, Carlsbad, CA, USA) from 500 ng of total RNA extracted from rice endosperm. Ampli Taq 360 (Applied Biosystems, Foster City, CA, USA) was used for subsequent PCR amplification with appropriate primers (Ramy1A-fwd, 5′-ATTGGGGTCTCAAGGAGGAG-3′; Ramy1A-rev, 5′-ATCGTGCGCTCAGATTTTCT-3′; Ramy3D-fwd, 5′-ATGGTGAAGATCGGGACGA-3′; Ramy3D-rev, 5′-CTTGAGCCCGCTATAGGTGC-3′). The Ubq primers were those of Miki and Shimamoto (2004). PCR consisted of initial denaturation (95 °C for 10 min) and 35 or 40 cycles of denaturation (95 °C for 30 s), annealing (55 °C for 30 s), extension (72 °C for 30 s) and final extension (72 °C for 10 min). Semi-quantitative RT–PCR was repeated once using different samples to confirm transcript levels.

RESULTS

Growth of rad mutant embryo during submergence

The growth of the rad mutant (as measured by the fresh and dry weights of the embryos) was not significantly different from that of the wild type after 1 d of submergence, but it was much less than that of the wild type between 3 and 7 d of submergence (Fig. 1). This finding agrees with previous studies on the rad mutant (Matsumura et al., 1995, 1998; Saika et al., 2006; Takahashi et al., 2011). The dissolved oxygen concentration in the water that we used was >0·188 mm at the start of treatment. Nevertheless, the growth response was similar in kind, though not in degree, to the response under anoxia (see Supplementary Data Figs S1, S2).

Fig. 1.

Fig. 1.

Open in a new tab

Growth of embryos of the wild type and rad mutant during submergence. Rice was germinated and grown during submergence for the indicated times. Fresh weights (A) and dry weights (B). Values are means (n = 15) ± SD. Asterisks indicate a significant difference between the wild type and rad mutant (P < 0·05, two samples, t-test).

In the embryos of dry seeds, in vitro ADH activities were 3·5-fold higher in the wild type than in rad (Fig. 2A). The ADH activity in the wild type embryos increased by 80 % at 1 d after imbibition and then remained at the same level, whereas the ADH activity in rad embryos remained at low levels until at least 7 d (Fig. 2A). The low ADH activity in the rad embryos during germination under submergence was due to a reduced level of ADH protein (Fig. 2C).

Fig. 2.

Fig. 2.

Open in a new tab

In vitro ADH activity in the embryos and endosperms, and ADH protein accumulation in the embryos of the wild type and rad mutant during submergence. Rice was germinated and grown during submergence for the indicated times. In vitro ADH activity was measured using total soluble proteins extracted from the embryos (A) or the endosperms (B). Western blotting of total soluble proteins extracted from the embryos was done using ADH-specific antibody (C; lower panel). Equal loading of total proteins was confirmed by SDS–PAGE (B; upper panel). Values are means (n = 3) ± SD. Asterisks indicate a significant difference between the wild type and rad mutant (P < 0·05, two samples, t-test).

The mutant tolerated 7 d of submergence as well as the wild type (Table 1). However, after 14 d of submergence, only 15 % of the mutant embryos recovered compared with 89 % for the wild type (Table 1). This indicates that low ADH1 activity in the rad mutant reduces not only growth, but also the long-term survival of the seedlings.

Table 1.

Viability of rice seedlings after submergence assessed by the leaf emergence after de-submergence

Strain 7 d submergence and 7 d de-submergence 14 d submergence and 7 d de-submergence
Wild type 95 ± 7 % 89 ± 2 %*
rad mutant 97 ± 6 % 15 ± 13 %*
Open in a new tab

Data are means of three replicates ± s.d.

In each replication, 20 seedlings were used for the experiment.

Asterisks indicate a significant difference between the the wild type and rad mutant (P < 0·01, two samples, t-test)

Ethanol production and lactate and alanine levels during submergence

Ethanol, which is produced by the submerged seedlings (i.e. by a combination of embryo and endosperm), was synthesized at about half the rate in mutant seedlings compared with that in wild-type seedlings (Table 2). Similarly, after 1 d of anoxia, ethanol production by the intact seedlings was significantly less in the mutant than in the wild type (Supplementary Data Table S1).

Table 2.

Ethanol concentration in the growth medium of rice seedlings

Strain Ethanol concentration in water (μm)
1 d 3 d 5 d 7 d
Wild type 13·7 ± 3·0 60·5 ± 5·5* 126·5 ± 8·1* 227·5 ± 40·9*
rad mutant 17·0 ± 13·5 22·8 ± 3·8* 63·2 ± 7·8* 110·4 ± 6·0*
Open in a new tab

The seedlings were in solution without forced turbulence and the escape of ethanol to the atmosphere was prevented by tight stoppers.

Data are means of three replicates ± s.d.

Asterisks indicate a significant difference between the wild type and rad mutant (P < 0·05, two samples, t-test).

Before submergence, the concentrations of lactate and alanine in the embryos of dry seeds were comparable between the wild type and rad mutant (Fig. 3). The lactate concentration in the rad embryos had dramatically increased by day 1 after submergence to 4·0 ± 0·67 µmol g−1 fresh weight (f. wt) and thereafter decreased, whereas the lactate concentration in the wild type decreased to negligible levels from an initial 1·1 ± 0·6 µmol g−1 f. wt for the 7 d of the experiment (Fig. 3). The alanine concentration in the wild type fluctuated between 4·5 ± 0·2 and 8·9 ± 1·1 µmol g−1 f. wt in the wild type, but increased to between 20·9 ± 1·8 and 23·2 ± 3·3 µmol g−1 f. wt in the rad mutant at 3–7 d of the experiment (Fig. 3).

Fig. 3.

Fig. 3.

Open in a new tab

Lactate and alanine concentrations in the embryos of the wild type and rad mutant during submergence. Rice was germinated and grown during submergence for the indicated times. Concentrations of l-lactate and l-alanine were measured using extracts from the embryos. Values are means (n = 3) ± SD. Asterisks indicate a significant difference between the wild type and rad mutant (P < 0·05, two samples, t-test).

Sugar concentrations in the embryo during submergence

Sucrose concentrations were similar between the wild type and rad embryo during submergence, except on day 5 when the concentration was higher in the mutant (Fig. 4A). In contrast, glucose and fructose concentration in the mutant were lower than in the wild type (Fig. 4B, C). At 3 d after submergence, the mutant embryo had 4·4- and 4·7-fold lower glucose and fructose concentration than the wild type at 3 d.

Fig. 4.

Fig. 4.

Open in a new tab

Concentrations of soluble sugars in the embryos of the wild type and rad mutant during submergence. Rice was germinated and grown during submergence for the indicated times. The concentrations of sucrose (A), glucose (B) and fructose (C) were measured using extracts from the embryos. Values are means (n = 3) ± SD. Asterisks indicate a significant difference between the wild type and rad mutant (P < 0·05, two samples, t-test).

Metabolic changes in the endosperm during submergence

The ADH activity in the endosperms, like ADH activity in the embryos, was much lower in the rad mutant than in the wild type during submergence (Fig. 2A, B).

Endosperm dry weight started to decrease after 3 d of submergence in the wild type, but not in the rad mutant (Fig. 5A). Starch contents showed a similar pattern (Fig. 5B), indicating that the decrease in dry weights of the wild-type endosperm was due to the decrease in starch. Similarly, under anoxia, the dry weight of the endosperm decreased less in the rad mutant than in the wild type (Supplementary Data Fig. S3).

Fig. 5.

Fig. 5.

Open in a new tab

Changes of dry weight and starch content in the endosperms of the wild type and rad mutant during submergence. Rice was germinated and grown during submergence for the indicated times. Endosperms were freeze-dried and the dry weights were measured (A). Starch contents were determined after solubilization of starch in the endosperms (B). Values are means (n = 3) ± SD. Asterisks indicate a significant difference between the wild type and rad mutant (P < 0·05, two samples, t-test).

Sucrose concentrations in the endosperm were comparable between the wild type and the mutant, as in the embryo (Fig. 6A). Glucose concentrations in the endosperm between 1 and 5 d were lower in the mutant than in the wild type and only converged at 7 d, due to decreases in the wild type and increases in the mutant (Fig. 6B). Fructose concentrations were very low and <0·1 mm in both the mutant and wild type. The levels of α-amylase isozymes RAMY1A and RAMY3D in the endosperm were much lower in the mutant than in the wild type (Fig. 7A). RAMY1A (α-amylase gene) mRNA, which is highly expressed during germination (e.g. Hwang et al., 1999; Loreti et al., 2003; Ismail et al., 2009), gradually increased after 1 d in the endosperm of both the wild type and mutant (Fig. 7B). RAMY3D expression could also be detected by increasing the number of PCR cycles. However, there was not a clear difference in RAMY3D levels between the wild type and rad mutant endosperm, except on day 0 (Fig. 7B).

Fig. 6.

Fig. 6.

Open in a new tab

Concentrations of soluble sugars in the endosperms of the wild type and rad mutant during submergence. Rice was germinated and grown during submergence for the indicated times. The concentrations of sucrose (A) and glucose (B) were measured using extracts from the endosperms. Values are means (n = 3) ± SD. Asterisks indicate a significant difference between the wild type and rad mutant (P < 0·05, two samples, t-test).

Fig. 7.

Fig. 7.

Open in a new tab

Protein accumulation of α-amylase (RAMY1A and RAMY3D) and mRNA accumulation of α-amylase (RAMY1A and RAMY3D) genes in the endosperms of the wild type and rad mutant during submergence. Rice was germinated and grown during submergence for the indicated times. Western blotting of total proteins extracted from the endosperms was done using RAMY1A- and RAMY3D-specific antibodies (A). Semi-quantitative RT–PCR of RAMY1A and RAMY3D mRNA was done using total RNA extracted from the endosperms (B). UBQ was used as a control.

The lower sugar concentrations in both embryo and endosperm raised the question of whether the response of the mutant seedlings would be improved by exogenous sugars, but this was not so for either 90 mm glucose or sucrose (Fig. 8).

Fig. 8.

Fig. 8

Open in a new tab

Germination and growth under submergence with external sugars. Wild-type and rad mutant seeds were germinated and grown under submergence with 90 mm glucose or sucrose for 3 d. Scale bar = 1 cm.

DISCUSSION

The main objective of this study was to assess how the low ADH activity in submerged rice affects (1) the metabolism in the embryo and (2) the ability of the seeds to transport sucrose from the endosperm to the embryo. To achieve this objective, it is necessary to consider to what extent submergence in semi-stagnant solutions resembles exposure to anoxia. The degree of oxygen deficiency would be determined by the inward oxygen diffusion from the water layer and oxygen consumption through metabolic activity in the cells (Armstrong and Beckett, 2011; Bailey-Serres et al., 2012). Hypoxia frequently occurs in the field (Colmer et al., 2011) and is more complicated than anoxia since, under hypoxia, some tissues are likely to have sufficient O2 for oxidative phosphorylation while other tissues called ‘cores’ may become anoxic (Berry and Norris, 1949). Therefore, the extensive literature on the metabolic responses to anoxia (Perata and Alpi, 1993; Bailey-Serres and Voesenek, 2008; Magneschi and Perata, 2009) is likely to be relevant to the metabolic responses to hypoxia. In the present study, the response of rice seedlings to submergence without forced turbulence was of the same kind, albeit different in degree, as the response to anoxia (Fig. 1; Supplementary Data Figs S1, S2). Specifically, elongation under submergence was confined to the coleoptile, as it is under anoxia, though the rate of elongation was about 3-fold greater in the semi-stagnant solutions than in anoxia at 3 d (Supplementary Data Fig. S1). This response under submergence is generally consistent with that described by Edwards et al. (2012), though Edwards et al. (2012) imposed hypoxia on rice seedlings by flushing with nitrogen gas containing 3 % O2. The flushing would have also caused turbulence. These results suggested that rice seedlings under our submerged conditions suffered from hypoxic stress.

During submergence, the ADH protein level and ADH activity were clearly lower in ADH1-deficient rad embryos and endosperm than in the wild type (Fig. 2). The low residual ADH activity in the rad mutant might be due to the activities of ADH2 and ADH3. The low ADH activity in the rad mutant appeared severely to suppress the growth of the embryo during submergence (Fig. 1) and to reduce survival when submergence lasted >7 d (Table 1). These results are consistent with the finding of a positive correlation between ethanol production and coleoptile elongation in rice during submergence (Kato-Noguchi, 2001) and during O2 deficiency and anoxia (Edwards et al., 2012). The growth and survival of the wild type during 14 d submergence is consistent with at least 5 d survival of excised rice coleoptile tips (Greenway et al., 2012; experiment terminated at 5 d anoxia)

In anaerobic rice coleoptiles, 92 % of pyruvate that is produced by glycolysis is converted to ethanol (Kato-Noguchi, 2006). The present experiments have clarified what happens to the alternative pathways of pyruvate consumption when, as in the rad mutant, ethanol formation is restricted. Reduced ADH activities in rice seedlings increased the lactate and alanine concentration under submergence (Fig. 3). These alternative end-products of glycolysis presumably accumulated since the flow of glycolytic intermediates to ethanol is restricted. However, the fact that the rad coleoptiles do not elongate under submergence suggests that this alternative route of pyruvate conversion is not enough to make up for the energy deficiency caused by low ethanol fermentation. An increase in the lactate concentration in the rad mutant might affect the cytoplasmic pH. Under hypoxia and anoxia, the cytoplasmic pH of young shoots of wheat (an anoxia-intolerant plant) can fall below that of rice shoots (an anoxia-tolerant plant; Menegus et al., 1991; Kulichikhin et al., 2007). Similarly to the reduced longevity of the rad mutant of rice, a maize adh1 mutant is highly sensitive to hypoxia and thus often dies within 48 h of hypoxic treatment (Lemke-Keyes and Sachs, 1989). During hypoxic treatment, the cytoplasmic pH in the root tip of the maize adh1 mutant continued to decrease (Roberts et al., 1984). Therefore, it would be of interest to measure the cytoplasmic pH in the rad mutant.

The low hexose levels in the embryo of the rad mutant raised the question of whether availability of substrate (i.e. glucose and fructose), rather than catabolism of sugars due to low ADH activities, was limiting the development of the rad mutant embryo. However limitation of carbohydrate catabolism rather than low ADH seems to be the case, since exogenous sugars did not improve development of the embryos (Fig. 8). In contrast, exogenous glucose improved both ethanol formation and growth in an anoxia-intolerant genotype (IR 22; Huang et al., 2003), so, in that case, metabolic activity in the endosperm is likely to be the limiting factor. The low hexose concentrations in the rad mutant embryo compared with the wild type are surprising, since sugar consumption in the rad mutant was clearly curtailed by less catabolism and lower consumption of carbohydrates in growth. The simplest explanation for the low hexose levels in the embryo would be that an energy deficit in the endosperm limited sucrose production and/or sucrose transport into the phloem. Starch hydrolysis might also be hampered by the low levels of amylases (Fig. 7A). The mRNA levels of α-amylase genes were not clearly different between the wild type and rad mutant (Fig. 7B), although the protein levels of α-amylase were clearly reduced in rad mutant endosperm (Fig. 7A). These results suggest that the low levels of amylases in the rad mutant could be due to the energy deficit depressing protein synthesis.

Sucrose concentrations were between 30 and 40 mm in both the mutant and wild-type endosperm. So, there was, presumably, enough substrate for the sucrose transporter in the endosperm of the rad mutant. Thus, it seems probable that an energy deficit during transport into the phloem is the likely cause of the reduced translocation. The rice sucrose transporter (SUT) transports sucrose from the endosperm to the embryo during germination (Scofield et al., 2007). The wheat scutellum has an apoplasmic barrier for solute movement (Aoki et al., 2006). Therefore, the sucrose transporter is important for sucrose transport from the endosperm to the embryo. Additionally, in rice seedlings, there is a steep sucrose concentration gradient between the endosperm (30–40 mm) and the phloem [220–300 mm (Taiz and Zeigler); 450–600 mm (Patrick, 2013)], so that energy is required to transport sucrose from the endosperm to the phloem against this gradient. Hence, sucrose needs to be transported against a steep free energy gradient of sucrose. Taken together, these results suggest that the ATP deficiency in the rad mutant impairs sucrose transport or starch degradation (as a result of impaired synthesis of α-amylase), leading to low glucose and fructose levels (Fig. 4B, C for sugar; Fig. 5 for starch).

The present investigation has shown that, even in a submerged environment containing substantial amounts of dissolved oxygen, a reduction in ADH (as brought about by an ADH1 mutation) reduces seedling viability, changes the balance between the end-products of glycolysis and decreases sugar concentrations in the endosperm and embryo. Exogenous sugar did not improve the growth or survival of the ADH1 mutant, indicating that sugar processing in the embryo was probably the limiting factor. However, how low ADH activity affects the endosperm deserves further experimental attention. The endosperm is well suited for investigations of sugar production and transport because of its simple composition and metabolism.

SUPPLEMENTARY DATA

Supplementary data are available online at www.aob.oxfordjournals.org and consist of the following. Methods: description of an experiment to determine the effects of an anoxic treatment of growth and ethanol production. Figure S1: coleoptile length during submergence and anoxia. Figure S2: growth of embryos of the wild type and rad mutant during anoxia. Figure S3: changes of dry weight in the wild type and rad mutant endosperms during anoxia. Table S1: ethanol production 1 day after anoxia.

Supplementary Data
supp_113_5_851__index.html (1,003B, html)

ACKNOWLEDGEMENTS

We gratefully acknowledge Timothy D. Colmer for teaching us the methods for ethanol measurement and for discussions. We thank Drs Kimiharu Ishizawa and Shin-ichi Arimura for stimulating discussions. This work was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists to H.T.

LITERATURE CITED

  1. Agarwal S, Kapoor A, Lakshmi OS, Grover A. Production and phynotypic analysis of rice transgenics with altered levels of pyruvate decarboxylase and alcohol dehydrogenase proteins. Plant Physiology and Biochemistry. 2007;45:637–646. doi: 10.1016/j.plaphy.2007.07.008. [DOI] [PubMed] [Google Scholar]
  2. Al-Ani A, Bruzau F, Raymond P, Saint-Ges V, Leblanc JC, Pradet A. Germination, respiration, and adenylate energy charge of seeds at various oxygen partial pressures. Plant Physiology. 1985;79:885–890. doi: 10.1104/pp.79.3.885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alpi A, Beevers H. Effect of O2 concentration on rice seedlings. Plant Physiology. 1983;71:30–34. doi: 10.1104/pp.71.1.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Aoki N, Scofield GN, Wang XD, Offler CE, Patrick JW, Furbank RT. Pathway of sugar transport in germinating wheat seeds. Plant Physiology. 2006;141:1255–1263. doi: 10.1104/pp.106.082719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ap Rees T, Jenkin LET, Smith AM, Wilson PM. The metabolism of flood tolerant plants. In: Crawford R.M.M, editor. Plant life in aquatic and amphibious habitats. Oxford: Blackwell Scientific; 1987. pp. 227–238. [Google Scholar]
  6. Armstrong W. Aeration in higher plants. Advances in Botanical Research. 1979;7:225–332. [Google Scholar]
  7. Armstrong W, Beckett PM. The respiratory down-regulation debate. New Phytologist. 2011;190:276–278. doi: 10.1111/j.1469-8137.2010.03537.x. [DOI] [PubMed] [Google Scholar]
  8. Atwell BJ, Greenway H. Carbohydorate metabolism of rice seedling grown in oxygen deficient conditions. Journal of Experimental Botany. 1987;38:466–478. [Google Scholar]
  9. Atwell BJ, Waters I, Greenway H. The effect of oxygen and turbulence on elongation of coleoptiles of submergence-tolerant and -intolerant rice cultivars. Journal of Experimental Botany. 1982;33:1030–1044. [Google Scholar]
  10. Bailey-Serres J, Voesenek LACJ. Flooding stress: acclimations and genetic diversity. Annual Review of Plant Biology. 2008;59:313–339. doi: 10.1146/annurev.arplant.59.032607.092752. [DOI] [PubMed] [Google Scholar]
  11. Bailey-Serres J, Fukao T, Gibbs DJ, et al. Making sense of low oxygen sensing. Trends in Plant Science. 2012;17:129–138. doi: 10.1016/j.tplants.2011.12.004. [DOI] [PubMed] [Google Scholar]
  12. Berry LJ, Norris WE. Studies of onion root respiration I. Velocity of oxygen consumption in different segments of root at different temperatures as a function of partial pressure of oxygen. Biochimica et Biophysica Acta. 1949;3:593–606. [Google Scholar]
  13. Beutler HO. Ethanol. In: Bergmeyer HU, editor. Methods of enzymatic analysis, vol VI. Metabolites. I. Carbohydrates. Basel: Verlag Chemie; 1983. pp. 598–606. [Google Scholar]
  14. Colmer TD, Huang S, Greenway H. Evidence for down-regulation of ethanolic fermentation and K+ effluxes in the coleoptile of rice seedlings during prolonged anoxia. Journal of Experimental Botany. 2001;52:1507–1517. doi: 10.1093/jexbot/52.360.1507. [DOI] [PubMed] [Google Scholar]
  15. Colmer TD, Winkel A, Pedersen O. A perspective on underwater photosynthesis in submerged terrestrial wetland plants. AoB Plants. 2011;2011 doi: 10.1093/aobpla/plr030. plr030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Davies DD. Anaerobic metabolism and the production of organic acids. In: Davies DD, editor. The biochemistry of plants. Vol. 2. London: Academic Press; 1980. pp. 581–611. [Google Scholar]
  17. Edwards JM, Roberts TH, Atwell BJ. Quantifying ATP turnover in anoxic coleoptiles of rice (Oryza sativa) demonstrates preferential allocation of energy to protein synthesis. Journal of Experimental Botany. 2012;63:4389–4402. doi: 10.1093/jxb/ers114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gibbs J, Greenway H. Mechanisms of anoxia tolerance in plants. I. Growth, survival and anaerobic catabolism. Functional Plant Biology. 2003;30:999–1036. doi: 10.1071/PP98095_ER. [DOI] [PubMed] [Google Scholar]
  19. Greenway H, Kulichikhin KY, Cawthray GR, Colmer TD. pH regulation in anoxic rice coleoptiles at pH 3·5: biochemical pHstats and net H+ influx in the absence and presence of NO3−. Journal of Experimental Botany. 2012;63:1969–1983. doi: 10.1093/jxb/err395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Guglielminetti L, Yamaguchi J, Perata P, Alpi A. Amylolytic activities in cereal seeds under aerobic and anaerobic conditions. Plant Physiology. 1995;109:1069–1076. doi: 10.1104/pp.109.3.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Huang S, Greenway H, Colmer TD. Anoxia tolerance in rice seedlings: exogenous glucose improves growth of an anoxia-‘intolerant’, but not of a ‘tolerant’ genotype. Journal of Experimental Botany. 2003;54:2363–2373. doi: 10.1093/jxb/erg252. [DOI] [PubMed] [Google Scholar]
  22. Hwang YS, Thomas BR, Rodriguez RL. Differential expression of rice α-amylase genes during seedling development under anoxia. Plant Molecular Biology. 1999;40:911–920. doi: 10.1023/a:1006241811136. [DOI] [PubMed] [Google Scholar]
  23. Ismail AM, Ella ES, Vergara GV, Mackill DJ. Mechanisms associated with tolerance to flooding during germination and early seedling growth in rice (Oryza sativa) Annals of Botany. 2009;103:197–209. doi: 10.1093/aob/mcn211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kadowaki K, Matsuoka M, Murai N, Harada K. Induction of two alcohol dehydrogenase polypeptides in rice roots during anaerobiosis. Plant Science. 1988;54:29–36. [Google Scholar]
  25. Kato-Noguchi H. Submergence tolerance and ethanolic fermentation in rice coleoptiles. Plant Production Science. 2001;4:62–65. [Google Scholar]
  26. Kato-Noguchi H. Pyruvate metabolism in rice coleoptiles under anaerobiosis. Plant Growth Regulation. 2006;50:41–46. [Google Scholar]
  27. Kulichikkhin KY, Aitio O, Chirkova TV, Fagerstedt KV. Effect of oxygen concentration on intracellular pH, glucose-6-phosphate and NTP content in rice (Oryza sativa) and wheat (Triticum aestivum) root tips: in vivo 31P-NMR study. Physiologia Plantarum. 2007;129:507–518. [Google Scholar]
  28. Lasanthi-Kudahettige R, Magneschi L, Loreti E, et al. Transcript profiling of the anoxic rice coleoptile. Plant Physiology. 2007;144:218–231. doi: 10.1104/pp.106.093997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lemke-Keyes CA, Sachs MM. Anaerobic tolerant null: a mutant that allows Adh1 nulls to survive anaerobic treatment. Journal of Heredity. 1989;80:316–319. doi: 10.1093/oxfordjournals.jhered.a110860. [DOI] [PubMed] [Google Scholar]
  30. Loreti E, Alpi A, Perata P. α-Amylase expression under anoxia in rice seedlings: an update. Russian Journal of Plant Physiology. 2003;50:737–742. [Google Scholar]
  31. Magneschi L, Perata P. Rice germination and seedling growth in the absence of oxygen. Annals of Botany. 2009;103:181–196. doi: 10.1093/aob/mcn121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Matsumura H, Takano T, Yoshida KT, Takeda G. A rice mutant lacking alcohol dehydorogenase. Breeding Science. 1995;45:365–367. [Google Scholar]
  33. Matsumura H, Takano T, Takeda G, Uchimiya H. Adh1 is transcriptionally active but its translational product is reduced in a rad mutant of rice (Oryza sativa L.), which is vulnerable to submergence stress. Theoretical and Applied Genetics. 1998;97:1197–1203. [Google Scholar]
  34. Menegus F, Cattaruzza L, Mattana M, Beffagna N, Ragg E. Response to anoxia in rice and wheat seedlings. Plant Physiology. 1991;95:760–767. doi: 10.1104/pp.95.3.760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Miki D, Shimamoto K. Simple RNAi vectors for stable and transient suppression of gene function in rice. Plant and Cell Physiology. 2004;45:490–495. doi: 10.1093/pcp/pch048. [DOI] [PubMed] [Google Scholar]
  36. Patrick JW. Does Don Fisher's high-pressure mainfold model account for phloem transport and resource partitioning? Frontiers in Plant Science. 2013;4:184. doi: 10.3389/fpls.2013.00184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Perata P, Alpi A. Plant responses to anaerobiosis. Plant Science. 1993;93:1–17. [Google Scholar]
  38. Perata P, Pozuetaromero J, Akazawa T, Yamaguchi J. Effect of anoxia on starch breakdown in rice and wheat seeds. Planta. 1992;188:611–618. doi: 10.1007/BF00197056. [DOI] [PubMed] [Google Scholar]
  39. Perata P, Geshi N, Yamaguchi J, Akazawa T. Effect of anoxia on the induction of α-amylase in cereal seeds. Planta. 1993;191:402–408. [Google Scholar]
  40. Perata P, Guglielminetti L, Alpi A. Mobilization of endosperm reserves in cereal seeds under anoxia. Annals of Botany. 1997;79:49–56. [Google Scholar]
  41. Rahman M, Grover A, Peacock WJ, Dennis ES, Ellis MH. Effects of manipulation of pyruvate decarboxylase and alcohol dehydrogenase levels on the submergence tolerance of rice. Australian Journal of Plant Physiology. 2001;28:1231–1241. [Google Scholar]
  42. Raymond P, Al-Ani A, Pradet A. ATP production by respiration and fermentation, and energy charge during aerobiosis and anaerobiosis in twelve fatty and starchy germinating seeds. Plant Physiology. 1985;79:879–884. doi: 10.1104/pp.79.3.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Roberts JKM, Callis J, Wemmer D, Walbot V, Jardetzky O. Mechanism of cytoplasmic pH regulation in hypoxic maize root tips and its role in survival under hypoxia. Proceedings of the National Academy of Sciences, USA. 1984;81:3379–3383. doi: 10.1073/pnas.81.11.3379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Saika H, Matsumura H, Takano T, Tsutsumi N, Nakazono M. A point mutation of Adh1 gene is involved in the repression of coleoptile elongation under submergence in rice. Breeding Science. 2006;56:69–74. [Google Scholar]
  45. Scofield GN, Aoki N, Hirose T, Takano M, Jenkins CLD, Furbank RT. The role of the sucrose transporter, OsSUT1, in germination and early seedling growth and development of rice plants. Journal of Experimental Botany. 2007;58:483–495. doi: 10.1093/jxb/erl217. [DOI] [PubMed] [Google Scholar]
  46. Taiz L, Zeigler E. Plant physiology. Redwood City, CA: The Benjamin Cummings Publishing Company, Inc; 1991. [Google Scholar]
  47. Takahashi H, Saika H, Matsumura H, Nagamura Y, Tsutsumi N, Nishizawa NK, Nakazono M. Cell division and cell elongation in the coleoptile of rice alcohol dehydrogenase 1-deficient mutant are reduced under complete submergence. Annals of Botany. 2011;108:253–261. doi: 10.1093/aob/mcr137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Terada R, Johzuka-Hisatomi Y, Saitoh M, Asao H, Iida S. Gene targeting by homologous recombination as a biotechnological tool for rice functional genomics. Plant Physiology. 2007;144:846–856. doi: 10.1104/pp.107.095992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wiengweera A, Greenway H, Thomson CJ. The use of agar nutrient solution to stimulate lack of convection in waterlogged soils. Annals of Botany. 1997;80:115–123. [Google Scholar]
  50. Xie Y, Wu R. Rice alcohol-dehydrogenase genes: anaerobic induction, organ specific expression and characterization of cDNA clones. Plant Molecular Biology. 1989;13:53–68. doi: 10.1007/BF00027335. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Data
supp_113_5_851__index.html (1,003B, html)
supp_mct305_mct305supp.pdf (118.9KB, pdf)

Articles from Annals of Botany are provided here courtesy of Oxford University Press

RESOURCES