Planta (Bed.) 75, 253--274 (1967)

Effects of Low Water Potentials on Respiration and on Glucose and Acetate Uptake, by Chlorella pyrenoidosa H. G ~ E ~ w A u and R. G. H I L L ~ Irrigation Research Laboratory, C.S.I.R.O., Griffith, N.S.W. University of Nottingham Department of Agricultural Sciences, Sutton Bonington, Loughborough, U.K. Received April 6, 1967

Summary. Chlorella pyrenoidosa was subjected to a range of water potentials and the effects of these treatments on endogenous respiration and on the uptake and respiration of glucose and acetate were measured. For a given water potential the reductions were greatest for glucose, less for acetate, and least for endogenous respiration. At intermediate water potentials of about -- 10 atm, glucose respiration was depressed strongly at first, but this respiration approached control levels after two to three hours at low water potentials. The reduced respiration of substrates was caused by inhibition of glucose and acetate uptake, as demonstrated by laC uptake experiments over short periods. These effects on uptake are attributed to low water potentials, rather than to any possible competition between the molecules of the osmotica and the substrates. Evidence for this view includes the equal inhibitions of "glucose-induced" respiration by osmotica with such diverse molecular structure as mannitol, KC1, and polyethylene glycol 1540. More conclusively, glucose itself was used as an osmotic agent and its inhibition of "glucose-induced" respiration was very similar to that by mannitol solutions of equal water potentials. Respiratory activity was much less reduced than uptake. This was demonstrated by lowering the water potential of cells which had already absorbed glucose from a control medium. The subsequent respiration was much higher than that for cells continuously exposed to low water potential. The findings are discussed in relation to the reduced transport of ions and sucrose, which is known to occur in vascular plants subjected to a water stress. The results demonstrate the advantages of using a unicellular organism in the study of metabolic effects of water deficits in plants. Introduction Little is k n o w n concerning the effects of water shortage on the m e t a b o l i s m of plants. I t is generally believed (MoTH~S, 1956 ; KOZLOWSKr, 1964) t h a t h y d r o l y t i c reactions p r e d o m i n a t e a n d s y n t h e t i c reactions are depressed, b u t the evidence is c o n t r a d i c t o r y a n d unsatisfactory. I n t e r p r e t a t i o n of the effects of water deficit on changes of m e t a b o l i s m i n vascular p l a n t s is complicated b y the fact t h a t water deficits are rarely m e a s u r e d i n the p a r t i c u l a r tissues i n v e s t i g a t e d a n d b y indirect effects arising from v a r i a t i o n s i n s t o m a t a l opening, leaf t e m p e r a t u r e s , t r a n s p o r t

254

H. GREEICWAuand R. G. HILLER:

a n d l~fineral u p t a k e . Some of these complications are evaded b y using excised roots, tissue slices, w a t e r plants, a n d unicellular organisms. E x a m p l e s of this t y p e of approach can be f o u n d i n the work with Elodea eanadensis (WALTEI~, 1928) a n d Chlorella (Ft2CHTBAV~I~, 1957). F i n d i n g s o b t a i n e d with these tissues, or organisms, m u s t e v e n t u a l l y be tested on vascular plants. As previous findings differ so markedly, there is little p o i n t i n discussing t h e m a t this stage ; t h e y will be m e n t i o n e d later i n the appropriate context. I t was t h o u g h t t h a t a n y n e w exploration of the p r o b l e m should ab initio be w i t h a simple tissue. The single-celled green alga Chlorella pyrenoidosa is a c o n v e n i e n t subject, as m u c h is already k n o w n a b o u t its general m e t a b o l i s m a n d it can later also be used for photos y n t h e t i c studies. This paper describes e x p e r i m e n t s i n which cells were s u b j e c t e d to w a t e r deficits i n darkness a n d m e a s u r e m e n t s were made on endogenous respiration a n d on the respiration a n d u p t a k e of glucose a n d acetate. A second p a p e r will describe the i n c o r p o r a t i o n of radioactive c a r b o n i n t o various metabolic components. Methods

1. Cultural and Experimental Conditions a) Culture of Chlorella. Chlorella pyrenoidosa (Emerson strain) was cultured autotrophically in 250 ml sterile gas-washing bottles at 250 C. Illumination was provided by 4 100 watt tungsten filament bulbs at 15 cm from the bottles. The culture medium contained in m-equiv./1.: Is Ca++0.2, Mg++20, lqOa 12.2, H2PO ~ 7, and SO~- 20. Microelements were as p.p.m. : manganese 0.5, boron 0.5, zinc 0.05, copper 0.1, and molybdenum 0.1. Iron at 5 p.p.m, was in the EDTA form. The water potential of this medium is about --1.5 arm. The cultures received air with 4 per cent CO2, at a flow rate of 15 cm3 per rain. b) Condition* during Tests. Prior to the experiments, cells were harvested by centrifugation for 7 rain at 1500 g. After resuspension in the test medium, the cells were centrifuged again for 7 rain at 4000 g. The temperature during centrifugation was about 0 ~ C. The cells were then resuspended in test medium, containing 10 mM KH2POa and 0.1 mM Ca(NOn)2, with a pH of 4.7. The final cell concentrations during the experiments were between 1.5 and 2 per cent by volume. I n experiments on "glucose- and acetate-induced" respiration the substrates were usually supplied at 10 raM, but in some long-term experiments were at 20 raM. Cultures were harvested between 48 and 120 hr after inoculation. There was some indication that the age of the culture affected the degree of inhibition by the osmotica. However, no clear trends were apparent and in this paper the age of the cultures is quoted only in figures and tables. c) Water Potential Treatments. The water potential of the test medium is about --0.4 arm. Water potentials, varying between --5 and --20 arm, were imposed by addition of mannitol, glucose, purified polyethylene glycol (PG) 200, PG 1540, KCI and glycerine. The total water potential would be derived from the osmoticum, the buffer, and from the added substrates. To avoid confusion, the text and figures quote only

Effects of Low Water Potentials on Respiration

255

the water potentiMs of the osmotica and in the case of the control that of the buffer, i.e. --0.4 arm. d) Mannitol Penetration. saC mannitol at 5 mM was added to the cell suspension. Samples were taken in a similar manner to that for glucose uptake at 9, 60, and 180 min after mannitol addition. At 60 min a comparison was made between washing periods of 0.5, 1, 2, and 3 min.

2. Experimental Methods a) Respiration. Oxygen uptake was measured at 25 o C in Warburg flasks, and in the text will be referred to as respiration. Respiration was also measured as 14C02 evolution from cells which had been previously supplied with Na~14CO3 in the light for 41/3 hours. In these experiments 0.75 ml of packed cells received 16 ~mole of INa2s~CO3of specific activity 30 ~curies per ~mole. b) Uptake el Substrates. Glucose and acetate uptake were measured with 14C-glucose and acetate-2-s4C. After a period of exposure to the sac compounds, the cells were filtered on a standard grade, OXOID, membrane filter and washed for 30 seconds with deionised water. This washing increased the water potential suddenly, but was required to avoid streaking of the chromatograms by the large amounts of mannitol. I n the uptake experiments lasting 9 minutes, s4CO2 evolution never exceeded 0.5 per cent of the total s4C uptake. Thus the counts of the filtered cells were an adequate measure of the total uptake of substrates. c) Puri/ieation o/ Polyethylene Glycols and Radioactivity Measurements. Polyethylene glycol 200 and 1540 was purified by filtration through Amberlite IR45 and Zeo Karb 225 exchange columns. This purified PG contained less than 0.3 p. p.m. of aluminium and 0.002 p.p.m, of chromium and copper. After purification, water potentials were determined by Spanner psychrometers (SPA~ER, 1951). s4CO2 was absorbed in N KOH and precipitated as Ba14C03. All radioactivity measurements were with an end-window Geiger-Mfiller tube.

Results 1. Endogenous Respiration E n d o g e n o u s respiration was measured b y 0 2 u p t a k e i n W a r b u r g respirometers a n d b y 14C02 evolution a n d these m e t h o d s gave essentially similar results. A water p o t e n t i a l of - - 1 0 a r m did n o t reduce endogenous respiration a n d there was even a slight increase i n 0 2 u p t a k e . W a t e r p o t e n t i a l s of - - 2 0 arm, on the other h a n d , u s u a l l y s t r o n g l y suppressed b o t h O 2 u p t a k e a n d 14CO2 evolution (Figs. 1 a n d 2). Occasionally, endogenous respiration was n o t m a r k e d l y reduced a t - - 2 0 a t m (see for example the c a p t i o n to Fig. 4).

2. "Glucose- and Acetate-Induced" Respiration a) Mannitol as osmotieum. " G l u c o s e - i n d u c e d " respiration was reduced a t m u c h higher water potentials t h a n was endogenous respiration. R e d u c t i o n s occurred below - - 8 a r m m a n n i t o l a n d became v e r y p r o n o u n c e d a t - - 2 0 a r m (Figs. 3 A a n d B), w h e n the cells h a d v e r y similar rates of "glucose-induced" a n d endogenous respiration. Between

256

H. GEEENWAY and R. G. HILLER: Endogenous

Respiration

0,6

~

0.4

~

o.2

N o

0'

2030 "

5'o/o'

90

Time

10 ', I~0 150 '

190 '

220 '

in minutes o o-o.~ o - - - - - o - lo O-'"-O-2O

otm atm atm

T i.s.d, [ P = O . O S )

Fig. 1. Endogenous respiration in mannitol solutions of different water potentials (Expt. 1, 72-hour culture) 10,000 =\ ,lg

\\k'k\\

0

0 - 0"4 e t m

O

0-20

arm

5~000

0

0-0.4

C--,--.-O-

A

o

I 0

i 15

I 30

I 45

arm

IO a t m

l 75

I 105

! 135

Time

in

I l l 0 15

30

I 60

I 90

I 135

minutes

Fig. 2A and B. Evolution of 1~CO~from ceils which had absorbed H14CO~ prior to lowering their water potentials by addition of manni~ol. A At --10 arm (Expt. 2, 72-hour culture). B At --20 arm (Expt. 3, 72-hour culture) - - 9 and - - 1 3 arm, reductions were large during the early p a r t of the experiments, b u t respiration neared control levels after lengthy exposure to a water stress (Fig. 3 A). " A c e t a t e - i n d u c e d " respiration was decreased less t h a n glucose respiration. F o r example, acetate cespiration, although depressed b y

I 170

Effects of Low Water Potentials on ]~espiration

257

- - 2 0 a r m m an n it o l , was still distinct, whereas glucose respiration u n d e r t h e same conditions was negligible (Fig. 4B). b) Various Osmotica. A n u m b e r of osmotica were c o m p a r e d w i t h m a n n i t o l . A t - - 1 0 arm, reductions in "glucose i n d u c e d " respiration were Table 1. "Glucose-induced" respiration in di//erent osmotica. Rates in tel 0 2 per tel cells per hour A. At --10 atm (Expt. 7, 96-hr culture) Time in minutes

Control at --0.4 arm Mannitol Polyethylene Glycol 1540 KC1 Polyethylene Glycol 200 Glycerine

1.2 0.64 0.77 0.71 1.3 1.3

2.4 1.1 1.1 1.2 1.8 1.8

2.4 1.2 1.2 1.4 1.8 1.8

2.3 1.5 1.5 1.5 1.9 2.0

2.3 1.6 1.8 1.8 1.9 2.0

2.2 2.1 2.0 2.1 2.0 1.9

2.2 2.0 | 2.0 2.2 1.9 1.6

0.3

B. At --20 arm (Expt. 8, 96-hr culture) Time in minutes

| Control at --0.4 arm Mannitol Polyethylene Glycol 200 Glycerine Mannitol no glucose lKannitol at --20 arm and additional glycerine at 0.04 M

1.7 0.65 1.2 1.3 0.65 0.80

2.4 0.60 0.80 1.0 0.40 0.50

2.7 0.39 1.2 0.9 0.32 0.38

2.6 0.41 1.8 1.1 0.43 0.51

2.7 2.4 0.30 0.32 1.6 1.8 1.2 1.3 0.32 0.24 0.37 0.35

2.3 0.42 1.9 1.5 0.32 0.52

2.0 0.35 1.8 1.8 0.34 0.42

0.18 0.10 0.18 0.18 0.10 0.10

v e r y similar in KC1, P G 15401, a n d m a n n i t o l (Table 1 A). Glycerine an d P G 200 also r e d u c e d " g l u c o s e - i n d u c e d " respiration, b u t t h e reductions were smaller a n d r e s p i r a t io n rose r a p i d l y to control levels. I n t h e presence of glycerine a n d P G 200 a t a w a t e r p o t e n t i a l of - - 2 0 arm, " g l u c o s e - i n d u c e d " The results with PG 1540 reported in this paper were obtained with one particular stock solution of PG 1540. PG 1540 stock solutions prepared from different batches had a very severe inhibitive effect and these results will be reported in a separate communication.

258

H. GR~,~WAY and R. G. HILT,ER: Endogenous

and

Glucose

Respiration

Glucose

Respiration

|

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20 30

50

70

90

110

130

150

190

220

Time in minutes Glucose ~ -o.4 a t m D - - - . - D - Io a r m

Endogenous 0

o'-4

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Water

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9

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Time

in

Fig. 3A and B. "Glucose-induced" respiration in mannitol solutions of different water potentials. A At --10 arm and --20 atm (Expt. 1). Endogenous respiration was showz in Fig. 1 and that at --20 a~m is included in this figure for comparison. B At water potentials between --5 and --13 arm (Expt. 4, 72-hour culture) Endogenous,

Acetate

9

and

Glucose

Respiration

./'

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I 0

20

60

100

160

I

I

240

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266

in minutes

Time

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A -0.4 a t m

Z~'--'-~-10 L

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Fig. 4A and B. Comparison between acetate and glucose respiration in malmitol solutions at different water potentials. A "Acetate-induced" respiration at -- 10 a~d --20 arm (Expt. 5, 72-hour culture). B Comparison between acetate and glucose at --20 atm (Expt. 6, 96-hour culture). I n the experiment of Fig. 4B, very similar rates of respiration were obtained for endogenous respiration at --0.4 and for endogenous and "glucose-induced" respiration at --20 arm. The figure only presents the m e a n for these treatments

i

1~

Effects of Low Water Potentials on Respiration

259

respiration was initially greatly reduced b u t rose to substantial levels after the cells had been for some time at these v e r y low water potentials (Table 1B). This recovery was in contrast to mannitol treated cells, which had v e r y low rates of "glucose-induced" respiration t h r o u g h o u t the experiment (Table 1 B). Glucose

Resplrotion (Glucose or Mannitol as o s m o t i c u m )

J

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20

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130

170

200

Time

in m i n u t e s

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Fig. 5. The effect of glucose, used as an osmoticum at --10 and --20 arm on "glucose-induced" respiration (Expt. 9, 96-hour culture). Mannitol solutions of the same water potentials were used as a comparison and these solutions contained 0.01 M glucose Reductions in "glucose induced" respiration could be due to specific effects of the various osmotica, quite apart from the effects of low water potentials. This question was resolved b y using glucose itself as an osmotic agent at - - 1 0 and - - 2 0 a r m ; the resulting reductions in glucoseinduced respiration being v e r y similar to those induced b y mannitol solutions of equal water potentials (Fig. 5).

~

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..... i 190

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:Fig. 6A and B. Effect of lowering water potentials, following glucose ~bsorption from a control medium at --0.4 arm. A Addition of Polyethylene glycol 200 at --20 arm (Expt. 10, 96-hour culture). B Addition of P G 1540 at --10 arm (Expt. 11, 96-hour culture)

A

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=

t~

Effects of Low Water Potentials on Respiration

261

c) Respiration o/ Glucose Absorbed Prior to Lowering the Water Potentials. The decreased respiration at low water potentials might be due to a reduced activity of the respiratory pathways. If so, cells which had absorbed glucose from a control medium would be expected to show a rapid reduction of respiration following a lowering of water potentials. To test this polyethylene glycols were added to cells previously supplied with glucose for 40 to 50 rain. Respiration showed a steep, temporary rise immediately following addition of P G 200 at --20 a t m 1"5 dg

~

~

_

o-o.4 9-o - lO

arm atm

i I~ 0.5

"6 o

II 05

I 20

I 40 Time

I 60

I 80

,I 1oo

I ]40

in minutes

Fig. 7. Effects of mannitol at --10 arm on the respiration of glucose, which had been absorbed during a two-hour period preceding the lowering of water potentials (Expt. 12). The glucose was removed from the medium by spinning and washing the cells. After resuspension the cells received osmotica at time 0. The respiration rate in a glucose continued treatment was 2.3 F1 02 per ~xl of cells per hour (Fig. 6A). Subsequently, respiration dropped to control levels and then declined further below the rates existing prior to the reduction in water potential, tIowever, this decline was slow and for a considerable time the respiration was much higher than for cells which had been at low water potentials during the entire period of glucose supply (Fig. 6A). Very similar results were obtained with P G 1540 at --10 arm (Fig. 6B). I n another experiment, glucose was first removed from the medium by centrifugation and washing, before the water potential was reduced (Fig. 7). Subsequent respiration was equal for cells treated with control solutions and with mannitol at --10 arm. Thus, it seems unlikely t h a t a reduced activity of respiratory pathways was responsible for the low respiration, which occurred when glucose and osmotiea were supplied simultaneously (Fig. 3).

d) Respiration o/ Glucose added some time after lowering Water Potential. "Glucose-induced" respiration neared control levels some time

262

H. GREENWAuand R. G. HILLER:

after lowering the water potential (Fig. 3A) and this might be partly due to an adjustment of the cells to the solutions of high osmotic pressures. Alternatively, the increased respiration might be due to a gradual increase of metabolitcs derived from the glucose. This question was investigated b y subjecting the cells for some time to a low water potential Endogenous

and

Glucose

Respiration

.f,/.i

f[~"

2

/ L I

%\\~// A

0

I

80

I |20

I 150

I I I 190 210 230

~ 300 Time

,, I 390

O

I

I

I

]40 160 180

I

I

260 28O

B

in m i n u t e s

glucose 0

I

100

added

[]-0"4 arm

O - . - - . - - O - - - - - - - D - to o t t o

~ glucose added Glucose

added

Fig. 8. Respiration of glucose which was added some time after lowering the water potential (Expts. 13 and 14, 72-hour cultures)

before adding the glucose (Fig. 8). Glucose respiration was still depressed at first, but it approached control levels much more rapidly than when glucose and osmotica were added at the same time (compare Figs. 8 and 3).

3. Recovery alter Removal o/Osmotica I n water stress experiments, it is important to establish whether effects of a low water potential persist or disappear of after return to a

"

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I I I I 110 120130 150

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! I I 35 5060

and

I 235

then-o.4atm

I 200

hs.d.

0

II 05

( P == 0 " 0 5 )

removed

in m i n u t e s

~ Osmoticum

Time

I-

I 275

3

::i:i

[]

O~20

arm

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I I I | 110 120130 150

[]---4Z]---~-~0

I I I 35 ,5060

Respiration

and

I 235

then-0.4arm

I 200

I 275

'I~

Fig. 9. Effects of m a n n i t o l removal on "glucose-induced" respiration. T r e a t m e n t s in which t h e low water potentials were retained are shown ~or comparison (Expt. 15). The t r e a t e d cells were spun a t 6.000 r.p.m, a n d t h e n resuspended in control test medium. Spinning a n d resuspension of the control cells did not alter their respiration

0

s

4

}lucose

".0 T~

9

9

9

264

H. (~I~EEI~WAYand R. G. HLt~Ea:

4. Mannitol Penetration The results f r o m t h e m a n n i t o l p e n e t r a t i o n e x p e r i m e n t s , n o t p r e s e n t e d in detail, showed t h a t m u n n i t o l u p t a k e in Chlorella was small. A f t e r Cumulative plots

~0.4atm /~ 1:3--,----[-:Ima t m / / ~ D

D

--

/

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100

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40

80

I

I

I

130

160

190

Rote of oxygen consumptionl~./l~

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, - - -40~ " 1 80 0

,

B

190

~

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/

/

x-m

-.-~

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0'08

0

I

I

I

t

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4O

80

130

160

190

Time

in minutes

o

[3

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•

0-04

e

o I

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C

, 160

/

']

0"12

~

, 130

I 0

,

I I000 14

,

1

I

2000

3000

!

D

4000

C uptake (c.p. 1OOsec.)

Fig. 10A--D. Effects of low water potential, induced by marmi~ol, on glucose uptake over long periods and on the relationship between glucose uptake and respiration (Expt. 16, 96-hour cultures). A 14C-glucose uptake plotted cumulatively. B 14C02 evolution plotted cumulatively. C 02 consumption plotted as rates. D Relationship between ltC02 evolution and ltC glucose uptake 9 rain of mannitol treatment the concentration in the total cell volume was only 7 per cent of the external mannitol concentration. There was no further uptake between 9 and 180 min after mannito] addition, suggesting that the uptake was confined to the free space. However, mannitol

Effects of Low Water Potentials on Respiration

265

c o n t e n t s d i d n o t decrease when t h e washing p e r i o d was increased f r o m 30 to 180 seconds. A s s u m i n g t h a t m a n n i t o l p e n e t r a t e d t h e cells, osmotic a d j u s t m e n t was small, p r o v i d e d t h e m a n n i t o l was e q u a l l y d i s t r i b u t e d over t h e entire osmotic volume. A l t e r n a t i v e l y , m a n n i t o l u p t a k e was r e s t r i c t e d to certain cell c o m p a r t m e n t s a n d these w o u l d t h e n h a v e an i m p r o v e d osmotic adjustment. Table 2. Glucose uptake at low water potentials (expressed as a per cent of uptake at --0.4 arm). Glucose uptake was measured by labelling the medium with 14C-glucose. Glucose was at 0.01 M, unless stated otherwiese (A ) Glucose and mannitol added simultaneously

The glucose and mannitol were added at time 0 and the glucose uptake was measured by introducing high specific activity 14C-glucose at 40 and 150 minutes respectively (Expt. 19, 196-hr culture). Time after lowering the water potential

Uptake at -- 10 arm, as % of control

4 0 ~ 3 and 43--46

150--153 and 153--156

30

90

( B ) Glucose added some time after addition o] mannitol

Water potential

Water potentials lowered Water potentials lowered 15 rain before glucose addition 75 rain before glucose addition Glucose at 0.01 M Expt. 20* (48-hr culture) 67

--16 arm

Expt. 21 ** (72-hr culture) 75 1

--I0 arm

Expt. 22 ** (52-hr culture) 95

--10 arm

-- 10 arm * Means ** Means

Glucose at 16 ~M per litre Expt. 23 ** 9O of uptake measured at 6, 12, 18, and 25 rains after glucose addition. of uptake measured at 3, 6, 9, and 12 mins after glucose addition.

There was a small 14C02 evolution after 180 rain t r e a t m e n t w i t h 14C m a n n i t o l . H o w e v e r , this e v o l u t i o n was only 1.2 p e r cent of t h e 14C0~ e v o l u t i o n f r o m 1~C glucose o v e r t h e s a m e t i m e i n t e r v a l . Thus, t h e effects of a n y m a n n i t o l m e t a b o l i s m b y Chlorella are l i k e l y to insignificant, r e l a t i v e to t h e v e r y high s u p p l y of s u b s t r a t e s in m o s t e x p e r i m e n t s . 19 Planta (Berl.),Bd. 75

266

H. GREENWAYand 1%. G. HILLER:

5. Glucose and Acetate Uptake I t was shown earlier that respiratory activity following glucose uptake was retained at a high level even at --20 arm. This suggested that the reduction of glucose-induced respiration was due to an inhibition of substrate uptake. This possibility was investigated in the following experiments. I n long-term experiments, 14C-glucose uptake was reduced by -- 10 atm mannitol, and the time curve for uptake was similar to those for 14C02

Short

term

Long

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Figs. 11A u. B (for legends see p. 267)

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150

159

175

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Effects of Low Water Potentials on Respiration

267

e v o l u t i o n a n d o x y g e n c o n s u m p t i o n (Fig. 10A, B, a n d C). Moreover, t h e 14C0~ evolved r a t i o of ~4C glucose taken up was v e r y similar in t h e two t r e a t m e n t s , a n d d e p e n d e d m a i n l y on t h e t o t a l a m o u n t of laC-glucose t a k e n u p b y t h e cells (Fig. 10D). These results suggested t h a t glucose u p t a k e was inhibited, while glucose was r e s p i r e d r e a d i l y once i t was t a k e n up. This view was confirmed in a n u m b e r of e x p e r i m e n t s on glucose a n d a c e t a t e u p t a k e over s h o r t periods. M a n n i t o l a t - - 5 a r m d i d n o t reduce glucose u p t a k e , t h e u p t a k e s being 8050 a n d 8500 counts p e r 100 see p e r ~1 of cells over a 12 m i n u t e u p t a k e period, for - - 0 . 4 a n d - - 5 a r m r e s p e c t i v e l y . W h e n m a n n i t o l was a d d e d to lower t h e p o t e n t i a l t o - - 10 a r m t h e glucose u p t a k e was reduced, b o t h when s u b s t r a t e a n d t r a c e r glucose were a d d e d s i m u l t a n e o u s l y (Table 2B) a n d when t h e radioa c t i v e t r a c e r was a d d e d some t i m e after a d d i t i o n o f t h e s u b s t r a t e glucose (Table 2A). I n f u r t h e r e x p e r i m e n t s glucose u p t a k e was a g a i n r e d u c e d b y - - 10 a t m m a n n i t o l , b u t o n l y s h o r t l y a f t e r lowering t h e w a t e r p o t e n t i a l a n d n o t after p r o l o n g e d t r e a t m e n t a t - - 1 0 a r m (Fig. 11). M a n n i t o l a t - - 2 0 a r m i n d u c e d v e r y large r e d u c t i o n s in glucose u p t a k e , a n d this u p t a k e d i d n o t i m p r o v e a f t e r l e n g t h y exposure to low w a t e r p o t e n t i a l s (Fig. 11). A c e t a t e u p t a k e was r e d u c e d less t h a n glucose u p t a k e , for e x a m p l e a t - - 2 0 a r m glucose u p t a k e was negligible b u t a c e t a t e u p t a k e was s u b s t a n t i a l (Fig. 12A). All these effects on s u b s t r a t e u p t a k e were similar to those o b s e r v e d on t h e r e s p i r a t i o n i n d u c e d b y either glucose or acetate.

Fig. 11 A--D. Short-term glucose uptake at different times after imposing a water stress. The water potential was lowered first. Sometime thereafter, glucose uptake was measured by adding 0.01 M glucose labelled with 14C glucose, while continuing the water stress treatments. A and C Uptake measured between 15 and 40 minutes after lowering the water potential (Expt. 17, in Fig. A and Expt. 18 in Fig. C; both 96-hour cultures). B and D Uptake measured between 150 and 175 minutes after lowering the water potential (Expt. 17 and 18 respectively) The 14C0~ evolution (in c.p. 100 sec per ~1 of cells) at the end of the uptake period was as follows:

Control ~r at -- 10 arm Mannitol at --20 arm

Fig. 10A

Fig. 10B

Fig. 10C

Fig. 10D

15 7 4

14 8 --

18 6 --

15 16 --

Glucose uptake by the control was between 150 and 300 cp. 100 sec per 91 of cells. 19'

268

H. G~E~;WAY and 1~. G. HmL~:R:

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W a t e r potentials were lowered first, and 40 minutes later labelled acetate was added to the cultures. A Acetate and glucose uptake at low concentrations (Expt. 24, 96-hour culture). Acetate a t 52 ~Moles per litre and glucose at 16 ~Nfoles per litre. B Acetate at 0.002 M (Expt. 25, 48-hour culture). C Acetate a t 52 ~zMoles per litre (Expt. 26, 72-hour culture). D Acetate at high concentration of 0.002 M (Expt. 27)

D

Effects of Low Water Potentials on Respiration

269

Discussion The question arises to what extent Chlorella experienced a water deficit. Unicellular organisms reach equilibrium rapidly (DAINTY and HoP~, 1959) and then the cells would be at the same water potentials as the medium. Such reductions in water potential would be achieved b y decreases in water content, leading to decreases in turgor pressure at moderate potentials and to decreases in osmotic or matric potentials at very low water potentials. Further work is in progress to establish which of these processes contributed to the low internal water potentials of Chlorella. Severe water deficits have always the danger of permanent injury to the cells. For example, respiration was not only greatly reduced during water stress in Elodea canadensis, but these plants died after deplasmolysis (T~EBovx, 1903; WALTER, 1928). I n contrast, Chlorella was very resistant to low water potentials, as shown b y the complete recovery of "glucose-induced" respiration, after mannito] at --20 a r m was removed from the medium. This demonstrates t h a t the results in the presence of osmotica are related to reversible, physiological events and not to irreversible injury, or death, of a portion of the cells.

1. Respiration and Uptake o/ Glucose and Acetate "Glucose and acetate-induced" respiration was reduced to a greater extent than was endogenous respiration. Possibly, low water potentials reduced some reactions of the respiratory pathways, and this inhibition became apparent only when high respiration rates were induced b y feeding glucose or acetate. However, water stress did not induce accumulation of intermediates of the glycolytic p a t h w a y and TCA cycle, as measured after short-term feeding of xaC-ghicose or 14C-acetate (ttrLLSR and GR~E~WAu unpublished data). Furthermore, water-stressed and control cells evolved similar amounts of 14C02, provided these cells conrained the same amount of 14C derived from laC-ghicose (Fig. 10D). These observations indicate ready metabolism of glucose, once it is absorbed b y the cells. An alternative explanation for the reduced glucose and acetate respiration would be t h a t water stress inhibits the uptake of substrates. This view receives support because the inhibitions of respiration and uptake of substrates follow very similar patterns. T h a t is, these inhibitions are of the same order, and occur over the same periods, in the following cases : 1. Smaller reductions of both uptake and respiration for acetate than for glucose (Figs. 4 and 12A). 2. At --20 arm, both uptake and respiration of glucose are almost completely inhibited (Figs. 3, 4 and 11).

270

H. GRE~WAu and R. G. H m L ~ :

3. At -- 10 arm, glucose uptake and respiration are inhibited initially, but both these inhibitions disappear after some time of exposure to this water potential (Figs. 3 and 11). The above observations could still be interpreted by assuming t h a t respiration was reduced, in turn reducing uptake to the same extent. However, water potentials of --20 arm greatly reduced glucose uptake; even though high respiration rates were retained at the same potentials, when these were applied to cells which had already absorbed glucose from a medium at --0.4 arm (Fig. 6). This shows conclusively t h a t the inhibited glucose respiration is not due to a reduced activity of the overall respiratory pathways. I t has to be remembered t h a t this high respiration would be derived from a variety of sources, i.e. there still might be an inhibition of a metabolic step during the initial conversion of glucose. I n t h a t case one would expect accumulation of glucose in the cells, at least for some time. No such accumulation occurred and 14C glucose uptake was already reduced severely after an uptake period of only three minutes. Thus, the presented data strongly support the notion t h a t inhibitions of substrate uptake were the cause for the reduced respiration of glucose and acetate.

2. Causes/or Reduced Uptalce o/ Substrates Osmotiea might affect metabolism in other ways t h a n by lowering water potentials. Such non-osmotic effects merit particularly careful consideration for uptake of substrates and ions, because molecules of the osmotica m a y be much more concentrated near the uptake sites t h a n near reaction sites inside the cells. There are three possible mechanisms for t h e reduced uptake : 1. A blockage of pores in the cell walls, as suggested for polyethylene glycol (MA~scH~ER, SAXE~A, and MICEAEL, 1965). 2. A competitive inhibition. 3. A specific inhibition. None of these processes would be expected to give the very similar reductions in "glucose-induced" respiration, observed for such diverse molecules as KC1, P G 1540, and mannitol (Table 1). The above-mentioned inhibitions will now be considered in more detail. For possibilities 1 and 2 reductions in uptake should become more pronounced at low glucose and acetate concentrations. However, reductions in uptake were at least as pronounced at high as at low substrate concentrations (Figs. 11 and 12, Table 2). Similarly, respiration at --10 arm mannitol was not affected by variations in glucose concentration (Table 3). Moreover, in cases I and 2 one would expect that removal of the osmotica would rapidly abolish their competitive effect, but recovery at --20 arm was slow (Fig. 9). Thus, possibilities ] and 2 are very unlikely. In the ease of specific inhibition (possibility 3), pronounced effects would be expected even at low concentrations of the inhibitor. IIowever, marmitol concen-

Effects of Low Water Potentials on Respiration

271

Table 3. "Glucose induced" respiration in mannitol at --10 atm and with di]]erent glucose concentrations (Expt. 28, 72-hr culture). Values are expressed as a per cent o/the respiration rates o/the various glucose concentrations at --0.4 atm Time in minutes

Glucose concentration

5mM 10mM 50mM

10--50

50--70

70--215

215--280

38 42 31

28 30 24

118" 55 53

113" 100 70

* At 5 mM the glucose respiration of the control declined after 70 min, due no doubt to a depletion of the glucose supply. With this exception the rates of "glucose induced" respiration were very similar at all glucose concentrations. tration had to be raised to 0.38 M before substrate uptake was reduced. Moreover, at --10 arm both glucose uptake and respiration recovered in the presence of mannitol, and this is difficult to reconcile with a specific inhibition. The a b o v e a r g u m e n t s m a k e n o n - o s m o t i c effects v e r y unlikely. H o w e v e r , t h e m o s t convincing evidence was o b t a i n e d when glucose itself was used as a n o s m o t i c u m , because t h e n " g l u c o s e - i n d u c e d " r e s p i r a t i o n was r e d u c e d to a v e r y similar e x t e n t as in m a n n i t o l solutions of t h e same w a t e r p o t e n t i a l s (Fig. 5). Thus, i t has been d e m o n s t r a t e d quite clearly t h a t t h e r e d u c t i o n s in r e s p i r a t i o n a n d u p t a k e of s u b s t r a t e s were i n d u c e d b y t h e low w a t e r p o t e n t i a l s a n d n o t b y a n y specific effects of t h e osmotica. 3. Recovery during Treatment at Low Water Potentials

Glucose u p t a k e recovered to control levels, during a p r o l o n g e d t r e a t m e n t a t low w a t e r p o t e n t i a l s . One e x p l a n a t i o n w o u l d be t h a t i n t e r n a l r e s p i r a t o r y s u b s t r a t e s h a d increased g r a d u a l l y , t h u s increasing respiration. H o w e v e r , glucose u p t a k e also recovered when no glucose was s u p p l i e d during t h e first 21/2 hours a t low w a t e r p o t e n t i a l . T h e a l t e r n a t i v e e x p l a n a t i o n for t h e r e c o v e r y is a n osmotic a d j u s t m e n t of t h e cells. I n t h e case of m a n n i t o l t h e r e c o v e r y is confined to i n t e r m e d i a t e w a t e r potentials, which are only a few a r m lower t h a n w a t e r p o t e n t i a l s which d i d n o t reduce respiration. I n t h a t case t h e osmotic adj u s t m e n t m i g h t well be due to i n t e r n a l p r o d u c t i o n of solutes. Glycerine a n d P G 200 h a d less severe effects t h a n t h e o t h e r osmotica. Differences b e t w e e n glycerine a n d o t h e r osmotica h a v e also been f o u n d during seed germination, a n d in t h a t case a t t r i b u t e d to a high p e r m e a t i o n of glycerine (MANOgAR, 1966). 4:. E//eets on Endogenous Respiration

Tissues f r o m w a t e r - s t r e s s e d v a s c u l a r p l a n t s s o m e t i m e s show a n increased r e s p i r a t i o n (STool~]~l~, 1956). This i n c r e a s e d r e s p i r a t i o n m i g h t

272

H. GREENVCAYand R. G. HmT.~R:

be a direct effect of water stress, as indicated by the somewhat higher respiration at --10 atm than at --0.4 arm, in Chlorella. These increases are presumably very different in nature from the steep, temporary rises in endogenous respiration following application of KCI to beet discs (BENNET-CLARK and B E x o s , 1943), and of P G 200 to Chlorella which already had absorbed glucose from a control solution. In many other instances vascular plants show a decreased respiration (LsWT% 1958), and these decreases have been attributed to indirect, long-term effects of water stress on photosynthesis (Bnlx, 1962). However, direct effects of water stress are suggested by the reduced respiration of Chlorella. High electrolyte concentrations of 0.44 M also reduced oxygen uptake by synchronised cultures of Chlorella pyrenoidosa during most of the dark cycle, though 02 uptake was higher than the controls during the last two hours of the dark period (RIND and S o ~ D ~ , 1961). The reduced endogenous respiration is hard to reconcile with the high rates of "glucose-induced" respiration, which persisted after P G 200 at --20 atm was added to cells previously supplied with glucose. There are two alternatives: 1. The respiratory substrates were different, and some of the cycles involved in endogenous respiration were inhibited, while those for the substrates derived from recently absorbed glucose were not affected. 2. Endogenous respiration involved an uptake process. This is possible for a heterogeneous population of cells, when old cells might leak metabolites which then would be later absorbed and respired by younger cells. Such uptake would be reduced at low water potentials, hence reducing endogenous respiration. 5. Relevance to Water Stress in Vascular Plants Work with a unicellular organism is expedient, but its significance to to the physiology of water stress in vascular plants should always be considered. The reductions in glucose and acetate uptake of Chlorella are particularly relevant, because reductions in transport have also been found during water stress in vascular plants. Reductions in phosphorus uptake have been reported for tomatoes b y GATES (1957) and by L r ~ s ~ , MAYR, and CHWALA (1962). Water stress reduced transfer of assimilated 14C02 from the assimilating tissues to the vascular bundles in wheat (WxaDLAW, 1967) and translocation of 14C-sucrose in beans (PLAvT and R~rNHOT,1), 1965). Transport of the floral stimulus was also reduced by exposing the roots of Lolium temulentum to solutions of polyethylene glycol at --24 arm (HusAIN, 1967). Such results might be explained by reduced water flow in the case of ion transport and by changes in sink size during sucrose transloeation. However, a more direct effect of

Effects of Low Water Potentials on Respiration

273

water stress on transport mechanisms is implicated by the inhibition established here of glucose and acetate uptake by Chlorella. The causes for the inhibitions of uptake are unknown. Effects of external low water potentials could be due to internal low water potentials, or to concurrent changes in water content and turgor pressure. I n the present experiments, water potentials would remain low in the Chlorella cells throughout the experiments. Yet, both uptake and respiration recovered after lengthy treatment at low water potential and in some osmotica like glycerine and P G 200, recovery was quite rapid. These recoveries are presumably due to osmotic adjustment and then the reduced uptakes were caused by changes in water content or turgor pressure, rather than to low internal water potentials. Similarly, low external water potentials reduced the growth of A r e n a eoleoptiles through changes in turgor pressure, and not by the simultaneous reduction in internal water potentials (OaDx~, 1960). As for the physiological changes responsible for the inhibitions of uptake, one can only speculate. The surface area of outer cytoplasmic membranes might have been reduced, if so this would be similar to the destruction of plasmodesmata as described by LA~BE~TZ (1954). Alternatively, uptake per se was reduced, either by a reduced activity of the uptake mechanisms, or by increased leakage from the cells thus reducing any accumulation due to active uptake. The data presented in the present paper demonstrate that use of unicellular organisms can help considerably in understanding effects of water deficits. Particular advantage might arise from the use of synchronised cultures, because then effects of low water potentials on cells of different ages can be determined. The authors are indebted to Professor F. L. MILTHOR~E,University of Nottingham, for his stimulating interest during the work and preparation of the manuscript. We also received helpful criticisms from: Dr. BETTYKLEPrE~, C.S.I.R.O., GI~rFFIT~, N.S.W., Dr. C. B. OsMo~1), University of Cambridge, Dr. D. THO3~AS,University of Adelaide, S.A., and Mr. T. FLOWERS,University of Nottingham. Efficient technical assistance was rendered by Mr. J. E. QUIGLEu Department of Agricultural Chemistry, S~rTTO~BO~I~GTO~,and by Mr. P. H~;GHES,Irrigation Research Laboratory, C.S.I.t~.O., G~rFFIT~, N.S.W. 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., GlCIF~ITH,N. S. W. References BENNET-CLARK, T. A., and D. B~xo~: Water relations of plant cells. III. The respiration of plasmolysed tissues. New Phytol. 42, 65--92 (1943). B~ix, H. : The effect of water stress on the rates of photosynthesis and respiration in tomato plants and lob]olly pine seedlings. Physiol. Plantarum (Copenh.) 15, 10--20 (1962).

274

GREENWAYand ttILLV,~: Effects of Low Water Potentials on Respiration

DAINTY,J., and A. B. HorE: The water permeability of cells of Chara Australis R. Br. Aust. J. biol. Sci. 12, 136--145 (1959). Ft)CHTB~V~I~, W. : Trockenresistenzsteigerung nach osmotischer Adaptation bei Saccharomyces und Chlorella. Arch. Mikrobiol. 26, 209--230 (1957). G A ~ s , C. T.: The response of the young tomato plant to a brief period of watershortage III. Drifts in nitrogen and phosphorus. Aust. J. Biol. Sci. 10, 125--146 (1957). Hus~I~r I. : Water stress and apical morphogenesis in barley and Lolium temulenturn L. Ph. D. Thesis University of Adelaide, Adelaide, S.A. 1967. KOZLOWSKI, T. T. : Water metabolism in plants. New York: Harper and Row 1964. LA~BERTZ, P. : Untersuchungen fiber das Vorkommen yon Plasmadesmen in den Epidermisauftenw~nden. Planta (Berl.) 44, 147--190 (1954). LEVITT,J. : Frost, drought, and heat resistance. In: Protoplasmatologia, vol. VIII, p. 6. Wien: Springer 1958. LI~s~I~, H., H. H. MAYR, und C. CHWALA: Der Einflul3 des osmotischen Drucks einer Ns auf die Phosphoraufnahme durch Wurzeln yon Lycopersicon esculentum. Atompraxis 8, 4 - - 6 (1962). MA~co~t~, M. S.: Effect of "osmotic" systems on germination of peas (Pisum sativum). Planta (Berl.) 71, 81--86 (1966). MA~SCn~CER, H., M. C. SAXENA, n. G. ~ICHA~.: Aufnahme yon Phosphat durch Gerstenkeimpflanzen in Abh/~ngigkeit yore osmotischen Druck der N~hrlSsung Z. Pflanzenern~hr. Dfing. Bodenk. 105, 82--94 (1965). MOT~ES, K.: Der EinfluB des Wasserzustandes auf Fermentprozesse und Stoffumsatz. In: Handbuch der Pflanzenphysiologie, Bd. I I I , S. 656--664, herausgeg. v. H. BV~ST~6~. Berlin-GSttingen-Heidelberg: Springer 1956. O~DI~T, L. : Effect of water stress on cell wall metabolism of Avena coleoptile tissue. Plant Physiol. 35, 443--450 (1960). PLAVT, Z., and LEO~O~A REINHOLD: The effect of water stress on [~4C] sucrose transport in bean plants. Aust. J. biol. Sci. 18, 1143--1155 (1965). RIED, A., u. C. J. SOEDEI~: Wirkungen erh5hter Salzkonzentration auf den Gaswechsel synchron kultivierter Chlorella pyrenoidosa. Naturwissenschaften 48, 106--107 (1961). S r A ~ R , D. C. : The Peltier effect and its use in measurement of suction pressure. g. exp. Bot. 2, 145--168 (1951). STOCXER, O. : Die Diirreresistenz. In: Handbuch der Pflanzenphysiologie, Bd. III, S. 696--741, herausgeg, yon H. Bu~sT~5~. Berlin-GSttingen-Heidelberg: Springer 1956. T~EBOUX, 0.: Einige steffliche Einflfisse auf die Kohlens~ureassimilation bei submersen Pflanzen. Flora (Jena) 92, 49--76 (1903). W ~ T E ~ , It. : Die Bedeutung des Wassers~ttigungszustandes ffir die C02-Assimilation der Pflanzen. Ber. dtsch, bot. Ges. 46, 530--539 (1928). WARDLAW, I. F. : The effect of water stress on translocation in relation to photosynthesis and growth. I. Effect during grain development in wheat. Aust. J. biol. Sci. 20, 2 5 ~ 0 (1967). Dr. t~. G. HILLER Univ. of Nottingham, Dept. of Agricu]tural Sciences School of Agriculture Sutton Bonington, Loughborough, U.K.