Planta (Berl.) 78, 49--59 (1968)

Effects of Low Water Potentials on Some Aspects of Carbohydrate Metabolism in Chlorella pyrenoidosa R. G. HILI~I~ and H. GI~EE~WAY University of Nottingham, Department of Agricultural Sciences, School of Agriculture, Sutton Bonnington, Loughborongh, Great Britain Received August 21, 1967

Summary. Chlorellapyrenoidosa was subjected to low water potentials and the resulting changes in carbohydrate metabolism were measured. Water deficit reduced the incorporation of 14C-glucose into methanol insoluble compounds, principally starch and increased that into sucrose. Even moderate water deficit, for example potentials of --2.5 and --5 arm, greatly reduced the incorporation of 14C-glucose into uridiue diphosphate glucose, while laC levels of the hexose monophosphates changed little, indicating a direct stimulus of sucrose synthesis. This increased sucrose synthesis was one of the earliest effect of water deficit, because potentials of - 2 . 5 and --5 arm did not reduce respiration and glucose uptake. At lower water potentials (--10 arm or less) there was reduced 14C incorporation into all sugar phosphates. This resulted from a combination of reduced l~C-glueose uptake and increased sucrose synthesis. Water potentials as low as --20 arm had little effect on acetate uptake, or on the laC levels in the intermediates of the TCA cycle. This confirmed that low water potentials do not directly inhibit respiratory pathways in Chlorella. The results are discussed in relation to the effect of water deficit on levels of various metabolites in vascular plants, which have been reported by other workers. Introduction Little is known about the effects of low water potentials on the metabolism of plants. I t is generally believed that synthesis of macromolecules is reduced and hydrolysis accelerated (KozLowsxI, 1964). Most of the evidence for this view is derived from vascular plants, after lengthy exposure to low water potentials. Thus any effects on metabolism per se might have been confused with changes due to reduced photosynthesis and growth, or due to increased leaf temperatures caused by the reduced transpiration. Such complications are avoided in the present work by using a unicellular alga, Chlorella pyrenoidosa, in short term experiments in the dark. The effect of low water potentials on endogenous respiration, and on the u p t a k e a n d r e s p i r a t i o n of g l u c o s e a n d a c e t a t e , h a v e a l r e a d y b e e n described (G~EENWAY a n d HILLE~, 1967). T h e p r e s e n t p a p e r r e p o r t s effects of l o w w a t e r p o t e n t i a l s o n i n c o r p o r a t i o n of 1~C glucose i n t o s t a r c h , sucrose 4a Planta (Berl.), Bd. 78

50

t~. G. HILLER and g . GREE~WAY:

a n d i n t e r m e d i a t e s associated w i t h t h e glyeolytic p a t h w a y a n d on t h e i n c o r p o r a t i o n of acetate-2-~4C into i n t e r m e d i a t e s of t h e T C A cycle.

Methods Chlorella pyrenoidosa was cultured as previously described (GI,,E]~I~wAYand HILL]~I~, 1967). 2% v/v cell suspensions were exposed to low water potentials by the addition of m~nnitol and some time thereafter saC-glucose or acetate-2-14C were added. Exposure to these radioactive substrates varied between 3 and 200 min. The cells were then filtered on a standard grade, Oxoid, membrane filter, leached with cold deionised water and then killed rapidly in boiling 80 per cent methanol. Methanol insolubles were subsequently removed by a second filtering on an Oxoid filter. Two dimensional paper (Whatman No. 4) chromatograms were prepared by developing firstly in phenol saturated with water (100 gm phenol: 39.5 ml H oO) and secondly in n-butanol/acetic acid/water (74:19:50). Radioautographs were made from these chromatograms by exposure to "Ilfex" no-screen X-ray film and the 14C activity in the compounds thus localised determined by means of a thin end-window Geiger-Muller tube (Nuclear Chicago 1)47). The 14C in individual sugar monophosphates was determined after re-chromatography of the free sugars following incubation (in acetate buffer at pH 4.8) with a purified preparation of acid phosphatase (Schwartz "Polidase S"). 14C-glucose, NaH14COa and sodium acetate-2-14C were obtained from the Radiochemical Centre, Amersham. The following abbreviations are used in the text: G-I-P for glucose-1-phosphate, PGA for 3-phosphoglyceric acid, ATP for adenosine triphosphate, ADPG for adenosine diphosphate glucose, UDPG for uridine diphosphate glucose, and TCA cycle for tricarboxylic acid cycle.

a) E]/ects on 1~C Incorporation into Methanol Insoluble Compounds Low w a t e r p o t e n t i a l s decreased the f o r m a t i o n of m e t h a n o l insolubles. This was clearly d e m o n s t r a t e d at a w a t e r p o t e n t i a l of - - 5 a r m ; when i n c o r p o r a t i o n of 14C into m e t h a n o l insolubles was r e d u c e d quite m a r k e d l y , even t h o u g h this w a t e r p o t e n t i a l d i d n o t reduce t h e l~C-glucose u p t a k e (Fig. 1A). W a t e r p o t e n t i a l s lower t h a n - - 5 a t m reduce glucose u p t a k e (GI~2E~wAY a n d HILLER, 1967). This is also shown in Fig. 2 A for a long t e r m e x p e r i m e n t , with a continuous s u p p l y of ~aC-glucose. L o w w a t e r p o t e n t i a l s s t r o n g l y r e d u c e d t h e level of 1~C in t h e m e t h a n o l insolubles, p r e s u m a b l y in p a r t due to t h e concurrent r e d u c t i o n in glucose u p t a k e (Fig. 2A). Because of t h e t r e a t m e n t effects on glucose u p t a k e , t h e i n c o r p o r a t i o n in m e t h a n o l insolubles can be b e s t considered b y t h e r a t i o 1~C in methanol insolubles • I00 total ~4Cuptake by cells

(i.e. '4C in methanol insolubles as a % of the total activity). At both --0.4 and ~10 arm this ratio increased at first with time but later reached a steady value (Fig. 2B). However, the % of the total activity in methanol insolubles was lower at --I0 than at --0.4 atm (Fig. 2B:),

Effects of Low Water Potential on Carbohydrate Metabolism 1 A (HennitaFsetm)

51

1B(gannitol- 20arm) i

iI i

A

100

w

I .o.,

5O

r162

i

Z:r

o

1

i

i

691

o

3

i

Hinules o o tota114C uptake ] , z. ~ 1~C methanol insolubles ~ 9 incase o- - -o total ~"C uptake ~ .~o~ato ~ ' - " "~ 1~Cin methanol insolubles/ . . . . .

Fig. l A and B. Effects of low water potentials on the incorporation oI a4C-glueose and aeetate-2-z4C into methanol insolubles. All data arc mannitol treatments expressed as a percentage of the values in the controls (--0.4 arm). oo Total a4C uptake, ~ ~ 14C in methanol insolubles. A, l~C-g]ueose at --5 arm mannito]. 1~C methanol insolubles in the controls as a % of total t4C uptake was 12%, at all times. ]3, z4C-glueose and acetate-2-1~C ~t --20 arm mannitol, t4C meth~noI insolubles in the control as a % of total ~4C uptake was: 12% for glucose and 7% for acetate at all times

1~Cin ceils Control(-0.'4atrn) 1o~ Hannitel(-~0atm) Cin methanol hs01ubles 9,---,, Control(-O.uatm) / _m_m3000~--~Hannifal(-lOafm)/ ~ 60 2A ~.~//_~c ur

Control(-O.~nlrn) Nannital (-10atrn)

/-

2

2C

9- 4-0

~zooo g ~1000

fi 20

o 100 Minutes

200

0

i

10 Minutes

200

0

2000 ' 4060 Counts

Fig. 2 A - - C . Effects of low water potential (--10 arm) on the long term uptake of 0.01 M~C-glucose and its incorporation into methanol insoluble compounds. A, z4C uptake into cells and into methanol insolubles. 14C uptake , 9 Control (--0.4 arm), o - - o ~annitol (--10 arm); 14C in methanol insolubles * * Control ( - 0.4 arm), ~ ~ Mannitol (-- 10 atm). B and C, Percentage of totaU4C in methanol t4C in methanol insolubles insolubles, i.e. 14C uptake b y the cells X 100. , - - , Control (--0.4 arm), o 4*

o Mannitol (--10 arm)

52

]:~. G, I-IILLER a n d H. GREEI~WAY:

even when the cells contained the same amount of 14C derived from the laC-glueose (Fig. 2C). Thus there were direct effects of low water potentials on synthesis of methanol insolubles in addition to the indirect effects due to reduced glucose uptake. Short term experiments only were done at --20 arm and it was therefore impossible to separate the direct effects of this low water potential on incorporation into methanol insolubles, from the indirect effects due to reduced substrate uptake. The total uptake of 14C-glucose was strongly suppressed but there was however still some measurable ~4C incorporation into the methanol insolubles at this very low water potential (Fig'. 1 B). l~esults with acetate were quite different from those with glucose since the 14C incorporation into methanol insolubles derived from acetate-2-~4C was not affected even by potentials as low as --20 arm (Fig. 1 B).

b) 14C Incorporation into Methanol Soluble Compounds l~adioactive carbon was supplied as HI~C03 in the light and as 14C-glucose and14C-acetate in the dark. The effect of low water potentials was determined on the incorporation of 14C into intermediates of, or associated with, the glyeolytie pathway and of the TCA cycle. ~) Incorporation o/ 14C ]rom 14C-glucose. A comparison was made between the distribution of 14C, as measured at 3, 6 and 12 minutes after adding tracer amounts of 14C-glucose, in control cells and cells subjected to a water potential of --10 arm. I n Chlorella the 14C is mainly incorporated into sucrose, sugar phosphates and amino acids (Table 1). The amino acids reflect the levels of activity in the small pools of the corresponding keto-acids, since the two are probably in isotopic equilibrium via exchange transamination (HILLER and WAu~E~, 1961). I n both treatments the level of 14C in the sugar phosphates did not change greatly with time but there were increases in activity in sucrose and amino acids. Low water potentials greatly increased 14C sucrose synthesis, and this increase exceeded the concurrent reduction in ~aC methanol insolubles. All other intermediates contained less 14C at --10 than at --0.4 arm. This decrease was more marked in the sugar phosphates t h a n in the amino acids; for example at 12 rain the ~C content of the predominant amino acid, alanine, was not greatly depressed by --10 arm mannitol (Table l). Similar results were obtained from another experiment at a water potential of --10 arm, in which a sample was analysed 25 min after adding a tracer amount of 14C-glucose. This low water potential also increased the 14C content of sucrose, while decreasing that in sugar mono-

Effects of Low Water Potential on Carbohydrate Metabolism

53

Table 1. E]/ect o/ a water potential o/ --10 atm on the distribution o/ ~4C derived /tom laC-glucose. All data are counts/lO0 sec/td cells Control (--0.4 atm) Time in minutes Methanol insolubles Sucrose Monophosphates Diphosphates UDPG[ ADPGJ PGA Alanine Glutamate Aspartate Malate Fumarate] Total 14Cuptake

3

6

1,900 3,600 2,210 4,950 10,260 12,420 2,040 2,100

Mannitol (--10 atm) 12

3

7,200 10,400 13,380 2,810

430 7,420 4,010 900

2,410 520 6,910 2,410 2,650

1,310 220 490 -190

130 130 720 2 0 , 4 9 3 30,190 49,410

-14,870

2,533 320 830 -270

2,580 690 2,630 530 560

6

12

700 1,400 19,300 30,800 5,380 4,123 520 520 1,430 -1,230 230 540

1,130 -4,900 830 960

-130 29,330 43,663

In each treatment after 15 rain exposure to the stated water potential 2.5 ml of 2% v/v Chlorella received 5 fze 14C-glucose (Specific activity 123 me/raM). phosphates and methanol insoluble compounds. In this longer term experiment the increase in activity in sucrose equalled the decrease in the methanol insoluble compounds. As there is often a decrease in total laC-glucose uptake at a water potential of --10 arm the experiment of Table 1 was repeated at --2.5 and --5.0 arm except that a single sample after 9 rain was substituted for those at 6 and 12 rain. The results of the experiment at --5.0 arm are shown in Table 2. There is a marked increase in 14C incorporation into sucrose at --5.0 arm, whilst activity in U D P G and methanol insoluble compounds decreases and that in sugar monophosphates is not affected. Even at a water potential of --2.5 arm similar results were obtained. After 3 and 9 min the effect of treatment at this water potential was to decrease the 14C content of U D P G by 30% and 37% respectively and to increase that of sucrose by 30% at both times. The activity in other phosphorylated compounds was not affected. These results suggest that the primary effect of mannitol solutions of low water potential is a stimulation of the biosynthesis of sucrose. Qualitatively similar results were obtained in other experiments, with high levels of glucose (0.001 M) in mannitol solutions and with tracer amounts of 14C-glucose in solutions of polyethylene glycol (Carbowax 1540). The latter result suggests that effects described in this paper are due to low water potential and not specific for mannitol. Interpretation of the results with polyethylene glycol is however complicated 4b

P l a n t a (Berl.), Bd. 78

54

1%.G. HILLEI%and H. GREENWAY:

Table 2. EMect of a water potential o] --5 arm on the distribution o/ laC /rom 1aC-glueose. All data are counts/lO0 sec/#l cells Control (--0.4 arm) Time in minutes Methanol insoluble compounds Sucrose UDPG Glucose monophosphate Other monophosphates (mainly fructose) Sugar diphosphates PGA Other compounds Total l~Cuptake

3

9

2,465 5,050 4,455 12,600 3,460 6,180 3,110 8,350 1,838 2,210 195 995 361 630 3,680 5,635 19,570 41,650

1VIannitol (--5.0 atm) 3

9

1,965 3,450 7,260 20,070 1,240 2,376 3,450 8,370 935 2,421 490 1,854 379 945 3,081 8,200 18,800 47,685

After 15 rains pretreatment at the stated water potential 2 ml of a 1.7% v/v Chlorella suspension received 5 ~zcof 14C-glucose(Specific activity 123 mc/mM). by the toxicity of some aged preparations (G~E~NWAY and HILLE~, unpublished results) which results in greatly reduced 14C-glucose uptake compared to that in mannitol solutions of the same water potential. fi) Incorporation oI ~4C lrom HI~CO-3. After a supply of H~CO~ during a 4 hour light period, the cells were exposed to water potentials of --10 and --20 arm. Respiratory 14C02 evolution is depressed only at --20 arm under these conditions (G~]~ENwxu and H ~ L ] ~ , 1967). A water potential of --10 arm increased the 14C content of the methanol soluble compounds by about 20 % but did not greatly alter the activity in individual compounds, Table 3. Effect of low water potential (--10 arm) on the distribution of 14C previously incorporated by photosynthesis in I~C0~. l~esults are expressed as % of total activity in the methanol soluble/faction Control at start of water potential treatment Sucrose 60.9 Sugar phosphates 4.9 Aspartate 5.0 Glutamate 21.7 Alanine 4.8 Total 1~Cin methanol soluble 160,000 compounds (counts/10Osec/v1 cells)

Control after 1 hour

Mannitol (--10 arm) after 1 hour

64.5 2.8 4.4 22.0 3.7 160,000

70.2 4.3 3.4 15.6 4.8 190,000

Prior to this experiment 0.75 ml of packed cells (approx. 1% v/v suspension) received 16 ~mole of lqa2 1~CO~ (Specific activity 30 me/raM) and were exposed to light for 4 hours.

Effects of Low Water Potential on Carbohydrate Metabolism

55

a p a r t f r o m a s m a l l increase in mC sucrose (Table 3). A t - - 2 0 a r m ~here was a larger increase (55%) in a c t i v i t y of t h e t o t a l m e t h a n o l soluble c o m p o u n d s , m o s t of which was in sucrose. I n t e r p r e t a t i o n of this r e s u l t is c o m p l i c a t e d b y t h e depression in laCO2 outpu~ a l r e a d y noted. T h u s effects of low w a t e r p o t e n t i a l on 14C d i s t r i b u t i o n from H14CO~ are n o t so m a r k e d as those on 14C from 14C-glucose, b u t do i n d i c a t e a r e d i s t r i b u t i o n of sac m a i n l y into sucrose. Table 4. E//ect o/a water potential o[ --20 atm on the distribution of 1~C derived/rom acetate-2J~C. All data are expressed as % o/total activity in the methanol soluble ]faction Control (--0.4 atm) Time in minutes 3

Mannitol (--20 arm)

6

9

3

6

9

Glutamate

78.1

72.9

74.5

81.3

77.7

76.4

Asl0artate

7.7

9.6

6.0

9.2

9.9

11.4

3.1 7.8 3.3 20,400

4.3 13.2 -27,000

3.9 9.8 2.8 37,500

Glutamine Acids of TCA cycle Others Total 14C uptake (counts/]00 see/~zlcells)

5.4 6.4 2.3 5.9 --15,350 25,600

7.1 5.1 -32,500

In each treatment 2.5 ml Chlorella suspension received 5 pc sodium acetate-2-14C (Specific activity 38 me/raM). y ) Incorporation o/ 14C /rom Acetate-2-1aC. A w a t e r p o t e n t i a l of - - 2 0 a r m has little effect on t h e u p t a k e of a c e t a t e - 2 -14 C (G~I~E~WAr a n d I-IzLL]~, 1967). T a b l e 4 shows t h a t - - 2 0 a r m m a n n i t o l also has little effect on d i s t r i b u t i o n of 14C in i n d i v i d u a l m e t h a n o l soluble c o m p o u n d s , a l t h o u g h t h e r e are small increases in t h e 14C c o n t e n t of t h e a m i n o acids a n d g l u t a m i n e a t t h e expense of t h a t in acids of t h e T C A cycle. These results do n o t i n d i c a t e a n y blockage of t h e T C A cycle a t low w a t e r p o t e n t i a l s because t h e 14C from a c e t a t e has to pass t h r o u g h acids of t h e TCA cycle before entering a s p a r t a t e or g l u t a m a t e .

Discussion The results p r e s e n t e d 2-14C, in whole Chlorella - - 2 0 a r m do n o t i n h i b i t earlier conclusion, b a s e d

on t h e u p t a k e a n d 14C i n c o r p o r a t i o n of a c e t a t e cells, suggest t h a t w a t e r p o t e n t i a l s as low as r e a c t i o n s of t h e T C A cycle. This confirms our b o t h on endogenous r e s p i r a t i o n a n d on 14C02

output from radioactive glucose and acetate (GRE~NWAY 1967).

and H~LL]~,

BER~STEI~ (1961 and 1963) also expressed the view that low water potentials are unlikely to impair the reactions of the TCA cycle. This opinion was based on the fact that during isolation of the mitochondria low water potentials are required

56

1%.G. HrnLER and H. GI~EENWiu

in order to obtain optimum activity for many reactions. However, at high water potentials the mitochondria swell and burst, so that the low activity observed for some reactions under these conditions is possibly an artefact due to structural damage. Some mitochondrial reactions are reduced by low water potentials. For succinoxidase however, reduced activity is due to a lowering of the water potentials per se and occurs irrespective of the structural state of the mitochondria (Tvnm% 1954). In Chlorella low water potentials (--10 arm) increased the synthesis of i4C sucrose from 14C-glucose, while concurrent reductions of activity in other methanol soluble intermediates were more pronounced for sugar phosphates than for amino acids. Decreases in the level of sugar phosphates have been reported for Tri/olium subterraneum when under water deficit in soil (WILson and HUFFAXEI~, 1964). Such reductions in sugar phosphates might result from reduced carbohydrate phosphorylation due to ATP shortage (ZI~oLKEVlC~ and I~OGACHEVA,1954), or from increased phosphatase activity as shown for chloroplasts isolated from water stressed plants (NI~ and POLJAKOFF-MAYBE~, 1966). In keeping with the hypothesis that phosphorylation is reduced, WILSOI~ and HUFFAKER (1964) suggested that a reduced level of U D P G might be the cause for the reduced starch levels found in inany species when under water stress. However, in Chlorella there was a pronounced increase in sucrose synthesis which at short times usually exceeded the reduction in polysaceharide formation. This suggests that, in this case, the reduced activity in sugar phosphates results from their more rapid removal due to increased sucrose synthesis, rather than from the lack of ATP. The conclusion that ATP formation in Chlorella is not greatly affected at moderate water potentials is consistent with the recent observation that in beet leaves the formation of ATP is reduced only at exceedingly low water potentials (SANTA]~IUS, 1967). Accompanying the increased ~4C incorporation into sucrose is a decreased incorporation into the methanol insoluble compounds; principally polysaccharides consisting of polymerised glucose units (of. CASSELTO~ and SYI~ETT, 1962). Decreases in total polysaccharides and increases in sucrose, or in both mono- and disaccharides, have been reported for vascular plants, after exposure to low water potential for long periods (MOTIVES, 1956; ILJIN, 1957). Occasionally the decrease in polysaccharide content is not accompanied by a concurrent increase in sugar content (WooD~AMS and KOZLOWSKI, 1954; WADLEIGI-[ and AVERS, 1945). This reduction in polysaecharides has been attributed to reduced photosynthesis, increased polysaccharide hydrolysis and/or decreased synthesis (KozLowsI:I, 1964; MOTHES, 1956; WADLEIGK and Au 1945 and WOODI~AM and KOZLOWS]~I, 1954). However, most of the available data cannot easily be interpreted because they were obtained after long periods of water deficit.

Effects of Low Water Potential on C~rbohydrate Metabolism

57

The present results with Chlorella are of particular interest, since they occurred after only a brief time of water deficit (15 rain) and were observable within 3 rain of introducing 14C-glucose. In these shorb term experiments the hydrolysis of polysaccharides was not greatly affected by a low water potential, as shown by the similar levels of activity in methanol soluble compounds at --0.4 and --10 atm, in cells which had been previously supplied with H14C0~ in the light. On the other hand, the data demonstrate an immediate and pronounced suppression of polysaecharide synthesis. Some short term experiments by other workers favour the reduced synthesis hypothesis. For example, water deficit reduced the incorporation of laC into water insoluble compounds of sunflowers (PLAUT and OnDI~, 1964). Water deficit also reduced starch formation in leaves floating in the dark on glucose solutions, either when the leaves were wilted prior to transferance to the solutions (ToLL~AA~, 1925; I L J ~ , 1930), or when water deficit was imposed by sucrose solutions of different osmotic pressures (ILJ~, 1930). All these results might have been due to a reduced uptake of glucose by the individual leaf cells, rather than to their reduced ability to synthesise polysaccharides, a view first suggested by MOTHS~S (1956) on theoretical grounds. A reduction in glucose uptake at low water potentials has been recently demonstrated in Chlorella pyrenoidosa (Gms]~wAu and H r L L ~ , 1967). However, in the present work a more direct effect on polysaceharide formation was demonstrated, because even at --5 arm when glucose uptake was unaffected there were pronounced decreases in laC-glueose incorporation into methanol insolubles. The question arises whether the decreased starch formation and the increased sucrose synthesis are related and if so, which of these effects is the primary cause. The biosynthetic pathway of starch formation from glucose is the same in Chlorella as in higher plants and may be controlled at the step GIP-->ADPG by the level of inorganic phosphate and 3-phosphoglyeerie acid (SAccWALLand P~ElSS, 1967). Our results do not indicate any pronounced effect of low water potential on the laC content of phosphoglyceric acid, yet a change in this would be expected if the reduced starch formation was a direct effect. I t is more likely that the reduced starch formation resulted from an increased sucrose synthesis. The best evidence for this view was obtained at --5 arm, when the main decrease in 1~C activity was in UDPG, an immediate sucrose precursor, whereas the glucose monophosphates were unaffected. This result suggests a direct stimulation of sucrose synthesis. The increased synthesis of sucrose might be partially responsible for the recovery of Chlorella, after it has been exposed for some time to low water potentials (G~J~ENWAu and I t I L L ~ , 1967). Such osmotic adjustments are of importance in the adaptation of plants to an environment

58

R. G. Hn~L~ and H. GRv.~.~wxY:

of low w a t e r p o t e n t i a l . Moreover, t h e concurrent decrease in polysaccharide f o r m a t i o n could p o s s i b l y c o n t r i b u t e to t h e r e d u c e d cell extension k n o w n to occur in v a s c u l a r p l a n t s e x p o s e d to low w a t e r potentials. Thus t h e effects described in this p a p e r m e r i t f u r t h e r i n v e s t i g a t i o n a n d a n e l u c i d a t i o n a t t h e molecular level m i g h t p r o v i d e a basis for u n d e r s t a n d i n g t h e s t a r c h - s u g a r balance, as well as osmotic a d a p t a t i o n , b o t h in higher p l a n t s a n d in Chlorella. The authors are indebted to Professor F. L. MmT~ORP~ for his stimulating interest in this work and to Mr. J . E . QVIGL~Y, Department of Agricultural Chemistry, Sutton Bormington and Mr. P. H~G~Es, Irrigation Research Laboratory C.S.I.R.O., for their efficient technical assistance. One of the authors (H. G.) acknowledges receipt of a Nuffield Foundation travel grant and special leave by C.S.I.R.O. Part of the work was carried out at the Irrigation Research Laboratory, C.S.I.R,O., Griffith, I~.S.W. References B~,~sT~I~, L.: Osmotic adjustment of plants to saline media. I. Steady state. Amer. J. Bot. 48, 909--917 (1961). Osmotic adjustment of plants to saline media. II. Dynamic phase. Amer. J. Bot. 50, 360--370 (1963). CASS~LTO~,P. J., and P. J. S:e~V,TT: The oxidation of 14C-labelled glucose by Chlorella vulgaris. II. The f~te of radioactive carbon. Ann. Bot. 26, 83--94 (1962). G~]~E~wAY, It., and R. G. H~L~R: Effects of low water potentials on respiration and glucose and acetate uptake by Chlorella pyrenoidosa. Planta (Berl.) 75, 253--274 (1967). H~L~R, R. G., and D. A. W ~ L x ~ : Formation of labelled amino acids by exchange transamination. Biochem. J. 78, 56~60 (1961). ILxrN, W. S. : Der EinfluB des Welkens auf den Ab- und Anfbau der St~rke in der Pflanze. Planta (Berl.) 10, 170---184 (1930). Drought resistance in plants and physiological processes. Ann. Rev. Plant Physiol. 8, 257--274 (1957). KOZLOWSXI,T. T. : Water metabolism in plants. New York: Harper & Row 1964. MOT~ES, K.: Der EinfluB des Wasserzustandes auf Fermentprozesse und Stoffumsatz. Handbueh der Pflanzenphysiologie, Bd. III, S. 656--664, hrsgg, yon W. Rvm~A~D. Berlin-GSttingen-Heidelberg: Springer 1956. NI~,T., and A. POLJA~:o~-MAYB~a: The effect of water stress on activity of phosphatases from swiss chard chloroplasts. Israel J. Bot. 15, 12--16 (1966). PLAVT, Z., and L. ORDIX: The effect of moisture tension and nitrogen supply on cell wall metabolism of sunflower leaves. Physiol. Plant., Kobenhaven 17, 279--286 (1964). SA~TA~IVS,K . A . : ]:)as Verhalten yon COn-Assimilation, NADP- und PGSReduktion und ATP- Synthese intakter Blattzellen in Abh~ngigkeit vom Wassergehalt. Planta (Berl.) 73, 228--242 (1967). SA~WAT.,G. G., and J. P~EISS: Biosynthesis of starch in Chlorella pyrenoidosa. I. Regulation of ATP: c~-D-glucose-l-phosphate adenyl transferase (ADPGlucose Pyrophosphorylase) by Inorganic P and 3-PGA. Arch. Biochem. 119, 454---469 (1967). -

-

Effects of Low Water Potential on Carbohydrate Metabolism

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Dr. R. G. HILLER University of Nottingham, School of Agriculture Sutton Bormington Loughborough, Leicestershire

Effects of low water potentials on some aspects of carbohydrate metabolism in Chlorella pyrenoidosa.

Chlorella pyrenoidosa was subjected to low water potentials and the resulting changes in carbohydrate metabolism were measured.Water deficit reduced t...
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