- Email: [email protected]
Glucose metabolism in anaerobic rice seedlings
Plant Science, 45 (1986) 31--36
31
Elsevier Scientific Publishers Ireland Ltd.
GLUCOSE METABOLISM IN ANAEROBIC RICE SE E D L IN G S
ROBERT G. MAYNE* and HANS KENDE M S U D O E Plant Research Laboratory, Michigan State University, East Lansing, MI 48824 (U.S.A.)
(Received November 7th, 1985) (Revision received January 31st, 1986) (Accepted March 17th, 1986) More than 80% of the radioactivity from [U-~'C]glucose metabolised by anaerobic rice seedlings or by excised roots or coleoptiles was recovered as ethanol plus CO:; less than 5% was recovered as water-soluble acidic components. Rates of 1"CO2 formation from [U-l"C]glucose were similar in roots and coleoptiles in both N 2 and air atmospheres. More 1'CO2 was formed from [U-~4C]glucose than could be accounted for by ethanolic fermentation, and the specific yields of ~'CO: from [6-1'C]glucose and [1-~'C]glucose gave unusually high C-6/C-1 ratios (1.7) in the anaerobic coleoptile. The results may indicate that appreciable pentan synthesis occurs in the anaerobic coleoptile. Key words: anaerobiosis; fermentation (ethanol) ; glucose metabolism; Oryza sativa
Introduction Rice is the crop plant most tolerant of flooding and anaerobiosis; it is capable o f germination a n d growth even under severe hypoxia. Gr o wth of the m es oc ot yl and coleoptile is enhanced and r o o t and leaf growth is retarded b y lowered 02 concentrations, elevated CO 2 concentrations and the presence of ethylene [1], conditions which may exist when seedlings germinate unde r standing water. If seedlings are exposed to e xt r em e l y h y p 0 x ic atmospheres (e.g. 99.9% N2), only the coleoptile elongates (but w i t h o u t substantial cell-division) and growth o f the r o o t and first leaf is totally suppressed [2]. Fresh weight, dry weight and protein c o n t e n t of coleoptiles grown under N2 are depressed compared to aerobic controls [3]. In this sense, only the rice coleoptile appears truly tolerant o f anaerobiosis, even though its growth and d e v elo p men t are abnormal.
*Present address: Department of Pure and Applied Biology, Imperial College of Science and Technology, Prince Consort Road, London SW7 2AZ, U.K.
It has been suggested t hat the varying tolerance of higher plants to anaerobiosis has a direct metabolic basis, namely ferm ent at i on of c a r b o h y d r a t e to products o t h e r than ethanol [4]. Experimental support for this view is lacking, however; f e r m e n t a t i o n in a variety of flood-tolerant plants has been shown to be largely ethanolic [5--7] and accumulations of o t h e r putative end-products o f ferm ent at i on have n o t been shown t o confer any advantage during anaerobiosis. It is difficult to assess the adaptive significance o f alternative fermentive pathways by comparing the response to anoxia or flood tolerance of different species because of the different genetic backgrounds involved and because flood-related stresses ot her than h y p o x i a may also affect field performance. However, if differences in sugar metabolism are indeed the basis for the exceptional floodtolerance of rice, one might e x p e c t such differences to be more p r o n o u n c e d in rice coleoptiles than in rice roots, since the f o r m e r but n o t the latter elongate under e x t r e m e hypoxia. Accordingly, we have examined the metabolic fate o f [14C] glucose in anaerobic rice coleoptiles and roots to find o u t w h e t h e r fermentation proceeds differently in these two organs.
0168-9452/86/$03.50 © Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
32 Materials and methods
Seeds o f rice (Oryza sativa, cv. M9: Rice E x p e r i m e n t Station, Briggs, CA) were surfacesterilised and germinated in air with a minimum o f water (dark, 30°C). Plant material consisted o f 50--100 mg fresh wt. of coleoptiles or roots excised f r om 3
was sampled at the exit, and the O2 cont ent , as measured by gas c h r o m a t o g r a p h y , was found to be <0.1%. Evaporated ethanol was caught in a dry-ice cold trap and 14CO2 was trapped by bubbling the exhaust gas through a m i xt ure of Carbosorb and Permafluor (Packard Instruments, Downers Grove, IL); both trapping procedures were ~ 96% efficient. At the end of the incubation, the tissue was killed in 1.5 M HCIO4, and gas flow cont i nued for 1 h to collect any residual 14CO2 from the chamber. The tissue was homogenised in the incubation vial using a glass rod and the extract neutralised with KOH. Carrier ethanol (5 ml) was added, and a sample of ethanol was collected by distillation (1--2 ml in middistillation). The tissue was removed and reextracted in 80% ethanol, then dried and com bust ed in a sample oxidiser. The combined ethanolic extracts were dried down, the residue taken up in a m i xt ure o f H20 and CHC13 ( 5 : 1 ) , and the water-soluble c o m p o n e n t s separated by ion-exchange c h r o m a t o g r a p h y on D ow ex 50 (H ÷) and D ow ex 1 (HCOO-). In all cases, m ore than 95% o f the neutral radioactive substances co-chromatographed
Table I. End-products of metabolism of [U-~4C]glucose in aerobic and anaerobic rice seedlings. Radiolabelled glucose (0.5 mM) was supplied as described in the text. After 6 h, the distribution of radioactivity among the classes of compounds listed was assessed. Figures shown are specific yields (calculated as percentages of the amount of glucose metabolised during the incubation). The results were obtained in one experiment; two repeat experiments gave closely similar results.
Tissue :
Seedling
Root
Coleoptile
(endosperm removed) Atmosphere :
Air
N2
Air
N2
Air
N:
Glucose metabolised (% of supplied)
87
87
83
90
75
70
Insolubles CHCl3-soluble
39.6 0.3 18.9 0.4
42.0 42.3 3.6 2.2
28.4 2.5 21.4 0.4
39.3 44.5 8.2 2.6
32.5 0.5 10.8 0.8
35.7 53.0 3.6 0.9
Water-soluble Acidic Basic
14.0 19.7
3.0 2.7
25.9 20.9
2.2 2.2
39.8 8.7
3.0 2.9
% of metabolised ~4C found as: CO s Ethanol
33
100 "0
(D 0 u m
0 0 04
0
¢O m
0 50 0 V
"O
(D
Air----
N2
m
0 > Iii
N 2 -.- A i r
O4
O
¢O
2
'
,;,
6
8
Time (hours) Fig. 1. Time course of evolution of 1+CO~ in air and in N~ by excised roots and coleoptiles of rice seedlings supplied with [U-l+C]glucose. Tissues were preincubated for 1 h with 5 mM glucose in the initial flow-through atmosphere of air or N2 after which time (zero hours on the graph) radiolabelled glucose (5 raM, 0.5 uCi) was added using a hypodermic syringe. After 3 h (at the points arrowed) the atmosphere was changed from air to N 2 (closed symbols) or from N 2 to air (open symbols). 1+CO~ was collected and quantified as described in the text. In all cases, between 10--15% of the supplied radioactivity was recovered as 14CO2 by the end of the incubation. • o, coleoptile; • D, root.
34 with glucose on thin-layer chromatograms. Recovery of added radioactivity was 94% or better. Rates of '4CO2 evolution from [U-~4C]glucose (5 mM, 0.5 pCi) were measured by incubating the tissue and collecting 14CO2 as above, changing the solution in the CO2-trap every half-hour. Radioactivity was determined by liquid scintillation counting, and the results were corrected for quenching. Results and discussion Fermentation of glucose in anaerobic tissues of rice seedlings was essentially ethanolic; in seedlings and in excised organs, more than 80% of the radioactivity from [U-14C]glucose metabolised under anaerobiosis was recovered as [14C] ethanol plus '4CO2 (Table I). By contrast, acidic plus basic end-products comprised less than 6% of the anaerobically metabolised glucose, compared to 34--49% in air. Incorporation of radioactivity into ethanol-insoluble material was decreased by at least 60% in all tissues under anaerobic conditions. Incorporation into CHC13-soluble components was increased in N2, but comprised only 1--3% of the total metabolised radioactivity. The total amount of glucose metabolised during the incubation by all tissues was similar in air and in Ns. This reflected similar rates of glucose catabolism in air and in N2 as estimated by following rates of ~4CO2 evolution from [U-14C]glucose in appropriately treated excised roots and coleoptiles (Fig. 1). Tissues transferred from air to Ns showed no diminution of 14CO2 evolution; the converse transfer caused a slight increase in the rate of ~4COs production {Fig. 1), which, together with the lag observed after addition of the radiocarbon, may suggest that uptake of glucose under anaerobiosis limits its utilisation. As a comparison, barley seedlings were incubated under the same conditions for 6 h; the total amount of glucose metabolised was reduced by at least 90% under anaerobic conditions (data not shown). Figure 1 suggests the occurrence of a pro-
nounced Pasteur effect, and hence the presence in rice seedlings of a strong constitutive capacity for ethanolic fermentation. However, of the ~4COs evolved by anaerobic rice tissues, only 50--70% could be accounted for as originating from glycolysis and fermentation to ethanol, assuming 1 tool of CO2 released per mol of ethanol formed (Table I). The 14CO2 in excess of the expected amount comprised 10--20% of the total metabolised [U14C]glucose. Similar effects have been noted in intact anaerobic tissues not supplied with radiolabelled substrate; Bertani et al. [8] observed that during the first 6 h of anaerobiosis rates of COs evolution by rice seedlings were diminished relatively little and were substantially greater than rates of ethanol formation: the two rates eventually became equal after 12 h. The excess of 14CO2 was not caused by continuing activity of the tricarboxylic acid cycle under anaerobiosis; we supplied [U-~4C]ace tate to test this possibility, but found that less than 1% of the radioactivity was released as ~4CO2, compared to 23% in air. Nor was it due to the CO2-free N2 atmosphere affecting physiological decarboxylation reactions to produce artefactual release of ~4CO2; we performed an experiment in a 'stagnant' N2 atmosphere to which 5 tll/1 COs had been added (collecting all CO2 at the end of the incubation) and found 14CO2 and [14C] ethanol production to be similar to that shown in Table I. A third possible explanation is that glucose may be oxidised by the oxidative pentose phosphate pathway under anaerobic conditions, as has been suggested to occur in anaerobic seedlings of Echinochloa crus-galli [9]. Catabolism of [ U-~4C] glucose by this pathway would increase the amount of 14CO2 evolved per [14C] ethanol formed (though it should be noted that no mechanism has been established for the subsequent oxidation of the NADPH produced as a consequence). We therefore examined the catabolism of [ ~4C] glucose labelled at the C-1 and C-6 positions. In anaerobic tissues, 70% of the radioactivity from metabolised [1-~4C]glucose was found
35 Table II. End-products of metabolism of 0.5 mM [1-14C]glucose and [6-~4C]glucose in aerobic and anaerobic rice tissues. Figures shown are specific yields obtained in representative experiments. The C-6/C-1 ratio is [specific yield of 14CO~ from [6-14C]glucose]/[specific yield of ~4CO2 from [1-~4C]glucose]. Experimental conditions are described in Table I. Treatment ~4C-label
% of 14C metabolised found as:
CO:
Ethanol
Insol.
CHCl 3 " s ° l .
Water-soluble Acidic
Basic
Root
Air N:
C-1 C-6 C-6/C-1 . . . . C-1 C-6 C-6/C-1 . . . .
53.2 28.1 0.53 20.0 16.8 0.84
6.0 8.3
23.1 33.2
1.3 2.5
7.9 12.8
3.4 4.5
69.9 68.7
4.7 3.4
1.9 4.1
3.4 3.0
4.1 4.5
1.2 1.4
33.4 33.1
3.7 3.5
12.7 10.9
4.9 4.5
70.4 52.3
4.7 3.7
0.8 2.5
4.9 4.2
4.0 4.0
0.6 2.7
32.0 36.9
0.7 1.7
11.3 13.8
4.0 6.2
69.4 58.5
6.5 3.7
1.4 3.2
2.4 2.9
3.1 3.4
Coleoptile
Air N:
C-1 C-6 C-6/C-1 . . . . C-1 C-6 C-6/C-1 . . . .
41.8 39.7 0.95 20.1 34.3 1.71
Whole seedling
Air N2
C-1 C-6 C-6/C-1 . . . . C-1 C-6 C-6/C-1 . . . .
59.7 36.0 0.60 17.1 27.6 1.61
as [14C]ethanol and close to 20% as 14CO2 (Table II). A greater p r o p o r t i o n o f the radioactivity f r o m [6-~4C]glucose was r e c o v e r e d as 14CO2 in a n a e r o b i c coleoptiles and seedlings, t h o u g h [14C]ethanol still c o n s t i t u t e d the largest f r a c t i o n o f r e c o v e r e d radioactivity. T h e ratio o f the specific yields o f 14CO2 f r o m [614C]glucose and [1-14C]glucose (C-6/C-1 in CO2) [10] was increased in all a n a e r o b i c tissues c o m p a r e d with air c o n t r o l s , and rose t o values greater than 1.5 in the anaerobic c o l e o p t i l e and w h o l e seedling (Table II). Rates o f ~4CO: e v o l u t i o n f r o m tissues supplied with [1-~4C]glucose or [6-14C] glucose were linear over the first 8 h o f il-.cubation in air or N2. C a u t i o n m u s t be exercised in the interpret a t i o n o f C-6/C-1 ratios [ 1 0 ] , particularly in view o f the long i n c u b a t i o n times used here; h o w e v e r , it is clear t h a t o u r results c a n n o t be
e x p l a i n e d b y postulating t h a t glycolysis and t h e o x i d a t i v e p e n t o s e p h o s p h a t e p a t h w a y are the o n l y paths o f glucose m e t a b o l i s m u n d e r a n a e r o b i c conditions. A e r o b i c o x i d a t i o n o f glucose via glycolysis and the t r i c a r b o x y i i c acid cycle alone w o u l d be e x p e c t e d to give C-6/C-1 ratios o f 1.0. F l o w o f c a r b o n t h r o u g h the oxidative pentose phosphate pathway lowers this ratio, since t h e glucose entering the p a t h is d e c a r b o x y l a t e d at C-1 b u t n o t at C-6. Recycling o f the p r o d u c t s o f t h e o x i d a t i v e p e n t o s e p h o s p h a t e p a t h w a y back to h e x o s e and r e - e n t r y into the path m a y o c c u r [ 1 0 ] ; t h e e x t e n t o f this recycling d e p e n d s o n w h e t h e r the p a t h w a y is o f the F - t y p e o r t h e m o r e r e c e n t l y described L - t y p e [ 11 ]. Such recycling w o u l d raise t h e C-6/C-1 ratio f r o m a low value t o a m a x i m u m o f 1.0. U n d e r the a n a e r o b i c c o n d i t i o n s i m p o s e d here, m o s t o f the C-6 and
36 C-1 r a d i o c a r b o n f r o m glucose was e x p e c t e d t o be f o u n d in e t h a n o l ; h o w e v e r , a significant p r o p o r t i o n was evolved as 14CO2 and C-6/C-1 ratios were m u c h greater t h a n 1.0. T r i c a r b o x ylic acid cycle activity a p p e a r e d to be suppressed, and in a n y event w o u l d provide no e x p l a n a t i o n o f such a high ratio, even if t h e r e was limited activity u n d e r such e x t r e m e h y p o x i a ( < 0 . 1 % O2). R a n d o m i s a t i o n o f the labelled c a r b o n b y a n y o t h e r means which might o c c u r during long i n c u b a t i o n s might also raise a low ratio to a value a p p r o a c h i n g 1.0, b u t c a n n o t a c c o u n t for higher values. Glucose can also be m e t a b o l i s e d b y nontriose p a t h w a y s ; Stitt and ap Rees [12] have shown t h a t p e n t a n synthesis m a y c o n t r i b u t e greatly to 14CO2 release f r o m [6-14C]glucose in w h e a t leaves. This d e c a r b o x y l a t i o n o c c u r s during the c o n v e r s i o n o f UDP-glucuronic acid to UDP-xylose, and such a flow o f c a r b o n would allow C-6/C-1 ratios t o e x c e e d 1.0. H o w e v e r , if this were the case, a substantial a m o u n t o f r e c o v e r e d r a d i o a c t i v i t y f r o m [U'4C]glucose would be e x p e c t e d in a neutral, acidic or insoluble f r a c t i o n ; we did n o t observe this. It m a y have been t h a t p e n t a n s themselves were e v e n t u a l l y degraded and re-entered the g l y c o t y t i c and o x i d a t i v e p e n t o s e p h o s p h a t e p a t h w a y s , because o f the length o f the incub a t i o n period. In conclusion, we find t h a t rice seedlings have a large c o n s t i t u t i v e c a p a c i t y for ethanolic fermentation, accounting for 60--70% of glucose c a t a b o l i s m u n d e r N2. A strong Pasteur e f f e c t in the first few h o u r s o f anaerobiosis was i n d i c a t e d b y the u n d i m i n i s h e d rates o f CO2 e v o l u t i o n f r o m glucose.Whilst a significant p o r t i o n o f glucose m e t a b o l i s m c o u l d n o t be e x p l a i n e d by f e r m e n t a t i o n t o e t h a n o l and a n a e r o b i c r o o t s and coleoptiles a p p e a r e d to use glucose in d i f f e r e n t ways, this c a n n o t be c o n s t r u e d as evidence for alternative fermen-
tive p a t h w a y s {though these m a y a p p e a r during m o r e p r o l o n g e d a n a e r o b i c t r e a t m e n t ) . If, for e x a m p l e , p e n t a n synthesis is t h e cause o f the unusually high C-6/C-1 ratio observed in the a n a e r o b i c coleoptile, it m a y o n l y reflect a c a p a c i t y for elongation and synthesis o f new cell wall material, r a t h e r t h a n being an underlying m e t a b o l i c ' s t r a t e g y ' for survival u n d e r anoxia. Acknowledgements We t h a n k Dr. A n d r e w H a n s o n for his perceptive and helpful c o m m e n t s . This w o r k was s u p p o r t e d b y a grant f r o m the U.S. Departm e n t o f Energy, u n d e r C o n t r a c t No. DEAC02-76ER01338. References 1 I. Raskin and H. Kende, J. Plant Growth Regul., 2 (1983) 193. 2 H. Opik, J. Cell Sci., 12 (1973) 725. 3 A. Alpi and H. Beevers, Plant Physiol., 71 (1983) 30. 4 R.M.M. Crawford, Metabolic adaptation to anoxia, in: D.D. Hook and R.M.M. Crawford (Eds.), Plant Life in Anaerobic Environments, Ann Arbor Science, Michigan, 1978, p. 269. 5 P.N. Avadhani, H. Greenway, P. Lefroy and L. Prior, Aust. J. Plant Physiol., 5 (1976) 15. 6 A.M. Smith and T. ap Rees, Planta, 146 (1979) 327. 7 M.E. Rumpho and R.A. Kennedy, Plant Physiol., 68 (1981) 165. 8 A. Bertani, I. Brambilla and F. Menegus, J. Exp. Bot., 31 (1980) 325. 9 M.E. Rumpho and R.A. Kennedy, J. Exp. Bot., 34 (1983) 893. 10 T. ap Rees, Assessment of the contributions of metabolic pathways to plant respiration, in: D.D. Davies (Ed.), The Biochemistry of Plants, Vol. 2, Academic Press, London, New York, 1980, p. 1. 11 R.L. Heath, Plant Physiol., 75 (1984) 964. 12 M. Stitt and T. ap Rees, Phytochemistry, 18 (1978) 1905.
31
Elsevier Scientific Publishers Ireland Ltd.
GLUCOSE METABOLISM IN ANAEROBIC RICE SE E D L IN G S
ROBERT G. MAYNE* and HANS KENDE M S U D O E Plant Research Laboratory, Michigan State University, East Lansing, MI 48824 (U.S.A.)
(Received November 7th, 1985) (Revision received January 31st, 1986) (Accepted March 17th, 1986) More than 80% of the radioactivity from [U-~'C]glucose metabolised by anaerobic rice seedlings or by excised roots or coleoptiles was recovered as ethanol plus CO:; less than 5% was recovered as water-soluble acidic components. Rates of 1"CO2 formation from [U-l"C]glucose were similar in roots and coleoptiles in both N 2 and air atmospheres. More 1'CO2 was formed from [U-~4C]glucose than could be accounted for by ethanolic fermentation, and the specific yields of ~'CO: from [6-1'C]glucose and [1-~'C]glucose gave unusually high C-6/C-1 ratios (1.7) in the anaerobic coleoptile. The results may indicate that appreciable pentan synthesis occurs in the anaerobic coleoptile. Key words: anaerobiosis; fermentation (ethanol) ; glucose metabolism; Oryza sativa
Introduction Rice is the crop plant most tolerant of flooding and anaerobiosis; it is capable o f germination a n d growth even under severe hypoxia. Gr o wth of the m es oc ot yl and coleoptile is enhanced and r o o t and leaf growth is retarded b y lowered 02 concentrations, elevated CO 2 concentrations and the presence of ethylene [1], conditions which may exist when seedlings germinate unde r standing water. If seedlings are exposed to e xt r em e l y h y p 0 x ic atmospheres (e.g. 99.9% N2), only the coleoptile elongates (but w i t h o u t substantial cell-division) and growth o f the r o o t and first leaf is totally suppressed [2]. Fresh weight, dry weight and protein c o n t e n t of coleoptiles grown under N2 are depressed compared to aerobic controls [3]. In this sense, only the rice coleoptile appears truly tolerant o f anaerobiosis, even though its growth and d e v elo p men t are abnormal.
*Present address: Department of Pure and Applied Biology, Imperial College of Science and Technology, Prince Consort Road, London SW7 2AZ, U.K.
It has been suggested t hat the varying tolerance of higher plants to anaerobiosis has a direct metabolic basis, namely ferm ent at i on of c a r b o h y d r a t e to products o t h e r than ethanol [4]. Experimental support for this view is lacking, however; f e r m e n t a t i o n in a variety of flood-tolerant plants has been shown to be largely ethanolic [5--7] and accumulations of o t h e r putative end-products o f ferm ent at i on have n o t been shown t o confer any advantage during anaerobiosis. It is difficult to assess the adaptive significance o f alternative fermentive pathways by comparing the response to anoxia or flood tolerance of different species because of the different genetic backgrounds involved and because flood-related stresses ot her than h y p o x i a may also affect field performance. However, if differences in sugar metabolism are indeed the basis for the exceptional floodtolerance of rice, one might e x p e c t such differences to be more p r o n o u n c e d in rice coleoptiles than in rice roots, since the f o r m e r but n o t the latter elongate under e x t r e m e hypoxia. Accordingly, we have examined the metabolic fate o f [14C] glucose in anaerobic rice coleoptiles and roots to find o u t w h e t h e r fermentation proceeds differently in these two organs.
0168-9452/86/$03.50 © Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
32 Materials and methods
Seeds o f rice (Oryza sativa, cv. M9: Rice E x p e r i m e n t Station, Briggs, CA) were surfacesterilised and germinated in air with a minimum o f water (dark, 30°C). Plant material consisted o f 50--100 mg fresh wt. of coleoptiles or roots excised f r om 3
was sampled at the exit, and the O2 cont ent , as measured by gas c h r o m a t o g r a p h y , was found to be <0.1%. Evaporated ethanol was caught in a dry-ice cold trap and 14CO2 was trapped by bubbling the exhaust gas through a m i xt ure of Carbosorb and Permafluor (Packard Instruments, Downers Grove, IL); both trapping procedures were ~ 96% efficient. At the end of the incubation, the tissue was killed in 1.5 M HCIO4, and gas flow cont i nued for 1 h to collect any residual 14CO2 from the chamber. The tissue was homogenised in the incubation vial using a glass rod and the extract neutralised with KOH. Carrier ethanol (5 ml) was added, and a sample of ethanol was collected by distillation (1--2 ml in middistillation). The tissue was removed and reextracted in 80% ethanol, then dried and com bust ed in a sample oxidiser. The combined ethanolic extracts were dried down, the residue taken up in a m i xt ure o f H20 and CHC13 ( 5 : 1 ) , and the water-soluble c o m p o n e n t s separated by ion-exchange c h r o m a t o g r a p h y on D ow ex 50 (H ÷) and D ow ex 1 (HCOO-). In all cases, m ore than 95% o f the neutral radioactive substances co-chromatographed
Table I. End-products of metabolism of [U-~4C]glucose in aerobic and anaerobic rice seedlings. Radiolabelled glucose (0.5 mM) was supplied as described in the text. After 6 h, the distribution of radioactivity among the classes of compounds listed was assessed. Figures shown are specific yields (calculated as percentages of the amount of glucose metabolised during the incubation). The results were obtained in one experiment; two repeat experiments gave closely similar results.
Tissue :
Seedling
Root
Coleoptile
(endosperm removed) Atmosphere :
Air
N2
Air
N2
Air
N:
Glucose metabolised (% of supplied)
87
87
83
90
75
70
Insolubles CHCl3-soluble
39.6 0.3 18.9 0.4
42.0 42.3 3.6 2.2
28.4 2.5 21.4 0.4
39.3 44.5 8.2 2.6
32.5 0.5 10.8 0.8
35.7 53.0 3.6 0.9
Water-soluble Acidic Basic
14.0 19.7
3.0 2.7
25.9 20.9
2.2 2.2
39.8 8.7
3.0 2.9
% of metabolised ~4C found as: CO s Ethanol
33
100 "0
(D 0 u m
0 0 04
0
¢O m
0 50 0 V
"O
(D
Air----
N2
m
0 > Iii
N 2 -.- A i r
O4
O
¢O
2
'
,;,
6
8
Time (hours) Fig. 1. Time course of evolution of 1+CO~ in air and in N~ by excised roots and coleoptiles of rice seedlings supplied with [U-l+C]glucose. Tissues were preincubated for 1 h with 5 mM glucose in the initial flow-through atmosphere of air or N2 after which time (zero hours on the graph) radiolabelled glucose (5 raM, 0.5 uCi) was added using a hypodermic syringe. After 3 h (at the points arrowed) the atmosphere was changed from air to N 2 (closed symbols) or from N 2 to air (open symbols). 1+CO~ was collected and quantified as described in the text. In all cases, between 10--15% of the supplied radioactivity was recovered as 14CO2 by the end of the incubation. • o, coleoptile; • D, root.
34 with glucose on thin-layer chromatograms. Recovery of added radioactivity was 94% or better. Rates of '4CO2 evolution from [U-~4C]glucose (5 mM, 0.5 pCi) were measured by incubating the tissue and collecting 14CO2 as above, changing the solution in the CO2-trap every half-hour. Radioactivity was determined by liquid scintillation counting, and the results were corrected for quenching. Results and discussion Fermentation of glucose in anaerobic tissues of rice seedlings was essentially ethanolic; in seedlings and in excised organs, more than 80% of the radioactivity from [U-14C]glucose metabolised under anaerobiosis was recovered as [14C] ethanol plus '4CO2 (Table I). By contrast, acidic plus basic end-products comprised less than 6% of the anaerobically metabolised glucose, compared to 34--49% in air. Incorporation of radioactivity into ethanol-insoluble material was decreased by at least 60% in all tissues under anaerobic conditions. Incorporation into CHC13-soluble components was increased in N2, but comprised only 1--3% of the total metabolised radioactivity. The total amount of glucose metabolised during the incubation by all tissues was similar in air and in Ns. This reflected similar rates of glucose catabolism in air and in N2 as estimated by following rates of ~4CO2 evolution from [U-14C]glucose in appropriately treated excised roots and coleoptiles (Fig. 1). Tissues transferred from air to Ns showed no diminution of 14CO2 evolution; the converse transfer caused a slight increase in the rate of ~4COs production {Fig. 1), which, together with the lag observed after addition of the radiocarbon, may suggest that uptake of glucose under anaerobiosis limits its utilisation. As a comparison, barley seedlings were incubated under the same conditions for 6 h; the total amount of glucose metabolised was reduced by at least 90% under anaerobic conditions (data not shown). Figure 1 suggests the occurrence of a pro-
nounced Pasteur effect, and hence the presence in rice seedlings of a strong constitutive capacity for ethanolic fermentation. However, of the ~4COs evolved by anaerobic rice tissues, only 50--70% could be accounted for as originating from glycolysis and fermentation to ethanol, assuming 1 tool of CO2 released per mol of ethanol formed (Table I). The 14CO2 in excess of the expected amount comprised 10--20% of the total metabolised [U14C]glucose. Similar effects have been noted in intact anaerobic tissues not supplied with radiolabelled substrate; Bertani et al. [8] observed that during the first 6 h of anaerobiosis rates of COs evolution by rice seedlings were diminished relatively little and were substantially greater than rates of ethanol formation: the two rates eventually became equal after 12 h. The excess of 14CO2 was not caused by continuing activity of the tricarboxylic acid cycle under anaerobiosis; we supplied [U-~4C]ace tate to test this possibility, but found that less than 1% of the radioactivity was released as ~4CO2, compared to 23% in air. Nor was it due to the CO2-free N2 atmosphere affecting physiological decarboxylation reactions to produce artefactual release of ~4CO2; we performed an experiment in a 'stagnant' N2 atmosphere to which 5 tll/1 COs had been added (collecting all CO2 at the end of the incubation) and found 14CO2 and [14C] ethanol production to be similar to that shown in Table I. A third possible explanation is that glucose may be oxidised by the oxidative pentose phosphate pathway under anaerobic conditions, as has been suggested to occur in anaerobic seedlings of Echinochloa crus-galli [9]. Catabolism of [ U-~4C] glucose by this pathway would increase the amount of 14CO2 evolved per [14C] ethanol formed (though it should be noted that no mechanism has been established for the subsequent oxidation of the NADPH produced as a consequence). We therefore examined the catabolism of [ ~4C] glucose labelled at the C-1 and C-6 positions. In anaerobic tissues, 70% of the radioactivity from metabolised [1-~4C]glucose was found
35 Table II. End-products of metabolism of 0.5 mM [1-14C]glucose and [6-~4C]glucose in aerobic and anaerobic rice tissues. Figures shown are specific yields obtained in representative experiments. The C-6/C-1 ratio is [specific yield of 14CO~ from [6-14C]glucose]/[specific yield of ~4CO2 from [1-~4C]glucose]. Experimental conditions are described in Table I. Treatment ~4C-label
% of 14C metabolised found as:
CO:
Ethanol
Insol.
CHCl 3 " s ° l .
Water-soluble Acidic
Basic
Root
Air N:
C-1 C-6 C-6/C-1 . . . . C-1 C-6 C-6/C-1 . . . .
53.2 28.1 0.53 20.0 16.8 0.84
6.0 8.3
23.1 33.2
1.3 2.5
7.9 12.8
3.4 4.5
69.9 68.7
4.7 3.4
1.9 4.1
3.4 3.0
4.1 4.5
1.2 1.4
33.4 33.1
3.7 3.5
12.7 10.9
4.9 4.5
70.4 52.3
4.7 3.7
0.8 2.5
4.9 4.2
4.0 4.0
0.6 2.7
32.0 36.9
0.7 1.7
11.3 13.8
4.0 6.2
69.4 58.5
6.5 3.7
1.4 3.2
2.4 2.9
3.1 3.4
Coleoptile
Air N:
C-1 C-6 C-6/C-1 . . . . C-1 C-6 C-6/C-1 . . . .
41.8 39.7 0.95 20.1 34.3 1.71
Whole seedling
Air N2
C-1 C-6 C-6/C-1 . . . . C-1 C-6 C-6/C-1 . . . .
59.7 36.0 0.60 17.1 27.6 1.61
as [14C]ethanol and close to 20% as 14CO2 (Table II). A greater p r o p o r t i o n o f the radioactivity f r o m [6-~4C]glucose was r e c o v e r e d as 14CO2 in a n a e r o b i c coleoptiles and seedlings, t h o u g h [14C]ethanol still c o n s t i t u t e d the largest f r a c t i o n o f r e c o v e r e d radioactivity. T h e ratio o f the specific yields o f 14CO2 f r o m [614C]glucose and [1-14C]glucose (C-6/C-1 in CO2) [10] was increased in all a n a e r o b i c tissues c o m p a r e d with air c o n t r o l s , and rose t o values greater than 1.5 in the anaerobic c o l e o p t i l e and w h o l e seedling (Table II). Rates o f ~4CO: e v o l u t i o n f r o m tissues supplied with [1-~4C]glucose or [6-14C] glucose were linear over the first 8 h o f il-.cubation in air or N2. C a u t i o n m u s t be exercised in the interpret a t i o n o f C-6/C-1 ratios [ 1 0 ] , particularly in view o f the long i n c u b a t i o n times used here; h o w e v e r , it is clear t h a t o u r results c a n n o t be
e x p l a i n e d b y postulating t h a t glycolysis and t h e o x i d a t i v e p e n t o s e p h o s p h a t e p a t h w a y are the o n l y paths o f glucose m e t a b o l i s m u n d e r a n a e r o b i c conditions. A e r o b i c o x i d a t i o n o f glucose via glycolysis and the t r i c a r b o x y i i c acid cycle alone w o u l d be e x p e c t e d to give C-6/C-1 ratios o f 1.0. F l o w o f c a r b o n t h r o u g h the oxidative pentose phosphate pathway lowers this ratio, since t h e glucose entering the p a t h is d e c a r b o x y l a t e d at C-1 b u t n o t at C-6. Recycling o f the p r o d u c t s o f t h e o x i d a t i v e p e n t o s e p h o s p h a t e p a t h w a y back to h e x o s e and r e - e n t r y into the path m a y o c c u r [ 1 0 ] ; t h e e x t e n t o f this recycling d e p e n d s o n w h e t h e r the p a t h w a y is o f the F - t y p e o r t h e m o r e r e c e n t l y described L - t y p e [ 11 ]. Such recycling w o u l d raise t h e C-6/C-1 ratio f r o m a low value t o a m a x i m u m o f 1.0. U n d e r the a n a e r o b i c c o n d i t i o n s i m p o s e d here, m o s t o f the C-6 and
36 C-1 r a d i o c a r b o n f r o m glucose was e x p e c t e d t o be f o u n d in e t h a n o l ; h o w e v e r , a significant p r o p o r t i o n was evolved as 14CO2 and C-6/C-1 ratios were m u c h greater t h a n 1.0. T r i c a r b o x ylic acid cycle activity a p p e a r e d to be suppressed, and in a n y event w o u l d provide no e x p l a n a t i o n o f such a high ratio, even if t h e r e was limited activity u n d e r such e x t r e m e h y p o x i a ( < 0 . 1 % O2). R a n d o m i s a t i o n o f the labelled c a r b o n b y a n y o t h e r means which might o c c u r during long i n c u b a t i o n s might also raise a low ratio to a value a p p r o a c h i n g 1.0, b u t c a n n o t a c c o u n t for higher values. Glucose can also be m e t a b o l i s e d b y nontriose p a t h w a y s ; Stitt and ap Rees [12] have shown t h a t p e n t a n synthesis m a y c o n t r i b u t e greatly to 14CO2 release f r o m [6-14C]glucose in w h e a t leaves. This d e c a r b o x y l a t i o n o c c u r s during the c o n v e r s i o n o f UDP-glucuronic acid to UDP-xylose, and such a flow o f c a r b o n would allow C-6/C-1 ratios t o e x c e e d 1.0. H o w e v e r , if this were the case, a substantial a m o u n t o f r e c o v e r e d r a d i o a c t i v i t y f r o m [U'4C]glucose would be e x p e c t e d in a neutral, acidic or insoluble f r a c t i o n ; we did n o t observe this. It m a y have been t h a t p e n t a n s themselves were e v e n t u a l l y degraded and re-entered the g l y c o t y t i c and o x i d a t i v e p e n t o s e p h o s p h a t e p a t h w a y s , because o f the length o f the incub a t i o n period. In conclusion, we find t h a t rice seedlings have a large c o n s t i t u t i v e c a p a c i t y for ethanolic fermentation, accounting for 60--70% of glucose c a t a b o l i s m u n d e r N2. A strong Pasteur e f f e c t in the first few h o u r s o f anaerobiosis was i n d i c a t e d b y the u n d i m i n i s h e d rates o f CO2 e v o l u t i o n f r o m glucose.Whilst a significant p o r t i o n o f glucose m e t a b o l i s m c o u l d n o t be e x p l a i n e d by f e r m e n t a t i o n t o e t h a n o l and a n a e r o b i c r o o t s and coleoptiles a p p e a r e d to use glucose in d i f f e r e n t ways, this c a n n o t be c o n s t r u e d as evidence for alternative fermen-
tive p a t h w a y s {though these m a y a p p e a r during m o r e p r o l o n g e d a n a e r o b i c t r e a t m e n t ) . If, for e x a m p l e , p e n t a n synthesis is t h e cause o f the unusually high C-6/C-1 ratio observed in the a n a e r o b i c coleoptile, it m a y o n l y reflect a c a p a c i t y for elongation and synthesis o f new cell wall material, r a t h e r t h a n being an underlying m e t a b o l i c ' s t r a t e g y ' for survival u n d e r anoxia. Acknowledgements We t h a n k Dr. A n d r e w H a n s o n for his perceptive and helpful c o m m e n t s . This w o r k was s u p p o r t e d b y a grant f r o m the U.S. Departm e n t o f Energy, u n d e r C o n t r a c t No. DEAC02-76ER01338. References 1 I. Raskin and H. Kende, J. Plant Growth Regul., 2 (1983) 193. 2 H. Opik, J. Cell Sci., 12 (1973) 725. 3 A. Alpi and H. Beevers, Plant Physiol., 71 (1983) 30. 4 R.M.M. Crawford, Metabolic adaptation to anoxia, in: D.D. Hook and R.M.M. Crawford (Eds.), Plant Life in Anaerobic Environments, Ann Arbor Science, Michigan, 1978, p. 269. 5 P.N. Avadhani, H. Greenway, P. Lefroy and L. Prior, Aust. J. Plant Physiol., 5 (1976) 15. 6 A.M. Smith and T. ap Rees, Planta, 146 (1979) 327. 7 M.E. Rumpho and R.A. Kennedy, Plant Physiol., 68 (1981) 165. 8 A. Bertani, I. Brambilla and F. Menegus, J. Exp. Bot., 31 (1980) 325. 9 M.E. Rumpho and R.A. Kennedy, J. Exp. Bot., 34 (1983) 893. 10 T. ap Rees, Assessment of the contributions of metabolic pathways to plant respiration, in: D.D. Davies (Ed.), The Biochemistry of Plants, Vol. 2, Academic Press, London, New York, 1980, p. 1. 11 R.L. Heath, Plant Physiol., 75 (1984) 964. 12 M. Stitt and T. ap Rees, Phytochemistry, 18 (1978) 1905.