Planta (Berl.) 103, 302-309 (1972) 9 by Springer-Verlag 1972
The Effectiveness of Internal Oxygen Transport in a Mesophyte (Pisum sativum L.) M. T. H e a l y a n d W. A r m s t r o n g Department of Botany, University of Hull, England Received November 25 / December 20, 1971
Summary. The effectiveness of oxygen movement through pea seedlings has been assessed firstly by assaying for radial oxygen loss along the roots using the cylindrical Pt electrode technique, and secondly by measuring root growth in various oxygen-free media. I t was found that roots would grow in oxygen-flee 3 % agar to a length exceeding 20 cm, but when such plants were removed to oxygen-free 0.05% agar oxygen could not be detected in the apical segments in roots longer than 9.5 cm unless respiratory activity was curtailed by cooling. H the greater part of the root was retained in 3 % agar and only the apical region exposed to 0.05 % agar and assayed, oxygen loss always occurred. I t was concluded that the 3 % agar has a jacketing effect substantially reducing oxygen leakage from the root surface and thus allowing more oxygen to channel down to the apical regions. Root growth in unstirred air-saturated 0.05% agar matched the growth in oxygen-free 3% agar. Root growth in unstirred oxygen-free 0.05% agar was arrested at c. 9 cm. It is suggested that the effect produced by the aerated unstirred 0.05 % agar is consistent with a jacketing effect mitigating oxygen loss from the root and that a growth of 9 cm in unstirred deoxygenated agar is consistent with a smaller jacketing effect due to the unstirred medium. I t is proposed that the accumulation of respiratory tissue will eventually render inadequate any jacketing effect. X~urther aerobic development at this stage will require a supply of oxygen from the rooting medium. Introduction The possibility t h a t roots m a y o b t a i n oxygen from the atmosphere n o t o n l y via the soil b u t also i n t e r n a l l y via the aerial parts has been considered for some time. I n 1925, after c o m m e n t i n g on oxygen u p t a k e from the soil, C a n n o n wrote " I t is conceivable t h a t ... the m o v e m e n t of 02 m a y be i n the opposite direction, t h a t is to say, from the p l a n t i n t o a n d t h r o u g h the water-films of the soft a n d into the a t m o s p h e r e of the soil." Since t h e n evidence of i n t e r n a l O~ t r a n s p o r t has a c c u m u l a t e d a n d several methods have been used to o b t a i n this evidence. A n indirect m e t h o d employed is the oxidation of indicators a t the root surface i n a m e d i u m originally oxygen free (Leyton a n d Rousseau, 1957; Armstrong, 1967; Hook, Brown, a n d K o r m a n i k , 1971). Direct approaches
The Effectiveness of Internal Oxygen Transport
have been (a) the use of labelled O~ (Evans and Ebert, 1960; Barber, Ebert, and Evans, 1962; Jensen, Stolzy, and Letey, 1967) and (b) various techniques involving polarography (van der l:[eide, de BoerBolt, and van Raalte, 1963; Armstrong, 1964, 1967; Vartapetian, 1964; Greenwood, 1967). Using a cylindrical platinum electrode designed to encircle the root, Armstrong obtained a quantitative assay of O2 diffusing into an anaerobic medium from individual intact roots of wetland plants forming aerenchyma. He suggested that the internal transport of O 2 may be effective in supplying (i) the plants, respiratory needs and (ii) sufficient O 2 for oxidation of the rhizosphere thus enabling these plants to tolerate flooding. The capacity of wetland roots to buffer against the reducing conditions of the soil with a zone of oxygenation has been considered elsewhere (Armstrong, 1970). I n mesophytes, on the other hand, the potential of the normal intercellular space system to supply oxygen for root respiration and possible oxidation of the rhizosphere has been generally overlooked, soil oxygen having been considered as the only significant source of supply. However, Greenwood's data for oxygen transport in several vegetable seedlings have shown that oxygen passing via the aerial parts could supply sufficient oxygen for root respiration and elongation in oxygen-free media even in the absence of specialized air-space tissue (Greenwood, 1967a, b). Nevertheless the roots assayed were very short and this has been seriously overlooked by those seeking to extrapolate the results to root systems of more normal proportions (Crawford, 1969; Crawford and Tyler, 1969). I t is hoped that the following experiments will clarify the position. Materials and Methods Peas (Meteor) were surface sterilized with 1000 ppm HgC12, rinsed three times with sterile distilled water and germinated on damp tissue paper. They were grown on under conditions as described in the individual experiments.
Detection o/02 Movements Oxygen movements through the peas were studied by assaying for oxygen loss from the root surface using the polarographie cylindrical electrode technique. A detailed description of the apparatus, general theory and procedure for this technique is given elsewhere (Armstrong, 1967). In the present work an Ag/AgCI electrode immersed in saturated KCI in a polythene extension formed the anode, and a platinum cylinder (0.5 cm long, internal diameter 0.225 cm) the cathode. To ensure that the root passed centrally through the Pt electrode, guides were attached above and below the cylinder. The peas were secured with r roots in deoxygenated 0.05 % agar/water to which saturated KC1 (1 ml per 500 ml agarwater) had been added. Above the liquid the shoots were kept in a humid atmosphere to prevent excessive transpiration. After the residual oxygen in the liquid medium had been determined, the Pt electrode was drawn up round the root,
M. T. Healy and W. Armstrong:
leaving a shell of liquid between the root and the inner exposed surface of the electrode. A polarizing voltage applied to the electrodes leads to the reduction of O~ to water at the Pt electrode surface, a process involving a 4 electron transfer per molecule of O~ reduced. The amotmt of O3 reduced is measured as an electrical current in ySk and is then translated into oxygen flux (radial oxygen loss) as g • 10-Scm-2 root surface rain-1 (Armstrong, 1967). To determine the effect of root respiration on radial oxygen loss (R.O.L.) the following procedure was used. As a continuous recording of any oxygen flux from the root was made, the medium and immersed root were surrounded by an ice water mixture lowering the temperature from 23~ to 3~ which curtails respiration, and changes in the diffusion current were followed on a Goerz singlechannel recorder until equilibrium was reached. The final reading was then noted and converted to a value at 23~ (an increase of 25%). This conversion depends upon the observation that flux rates at 3~ are depressed by c. 20 % because of the increase in solubility and decrease in diffusion coefficient of oxygen at this temperature. The new 23~ value is therefore the flux rate which would be expected at that temperature with respiratory activity curtailed.
Results and Discussion
Experiment I Peas were g e r m i n a t e d i n sterile conditions a n d w h e n the radicles were e. 1.5 cm long the peas were placed o n 3 % agar gel with the radicle inserted i n t h e agar, a n d grown on a t 24~ i n a diffuse light. Oxygen diffusion rate (ODR) i n the m e d i u m proved to be zero, a n d c o n s e q u e n t l y the peas were deprived of a r e a d y s u p p l y of oxygen i n the rooting m e d i u m . U n d e r these conditions the m a i n root can readily grow i n excess of 20 cm. T h e e x t e n t of the root growth observed i n these experiments suggests t h a t oxygen diffused over distances m u c h greater t h a n those m e n t i o n e d i n Greenwood's papers. To ascertain if this was so, roots v a r y i n g from 6 to 20 cm were dissected from t h e agar a n d assayed for radial oxygen loss (R.O.L. / a t 1 cm i n t e r v a l s from apex to base. The results are given i n T a b l e I a n d Fig. 1. F a i l u r e to detect oxygen a t some p a r t s of the root surface (cont i n u o u s line, Fig. 1), could have been due to the i m p e r m e a b i l i t y of the root a t those sites. However, after cooling, 0 2 was detected a t all points along the root (broken line, Fig. 1), confirming t h a t the root wall was permeable a t all points assayed. This is consistent with the view t h a t r e d u c e d respiration leads to a rise i n 0 2 c o n c e n t r a t i o n w i t h i n the root a n d a s u b s e q u e n t increase i n flux from the root (Armstrong, 1971). F r o m the results o b t a i n e d it would seem too t h a t respiration h a d been m a k i n g considerable d e m a n d s o n the available oxygen supply. T a b l e I shows t h a t i n roots of u p to 9.5 cm oxygen diffused out of the root apex. I n longer roots R O L was detected a t v a r y i n g distances b e h i n d the apex, a l t h o u g h the diffusion p a t h t e n d e d to be longer i n
The Effectiveness of Internal Oxygen Transport
r= 6 1 . , .
Distance from root base (cm)
Fig. 1. R.0.L. along a pea root prior to ( o ~ ) , and after cooling (. . . . . ) Table 1 Root length (cm)
Distance from Apparent oxygen apex at which R0L diffusion path was first detected length (cm) (em)
4.0 5.0 5.5 6.3 7.0 7.5 8.0 8.5 9.5 10.0 10.0 11.5 11.5 12.5 15.5 18.5 18.5 21.5
0 0 0 0 0 0 0 0 0 2.5 2.0 3.0 1.5 2.5 7.5 7.5 7.5 6.5
4.0 5.0 5.5 6.3 7.0 7.5 8.0 8.5 9.5 7.5 8.0 8.5 10.0 10.0 8.0 11.0 11.0 14.0
longer roots. As no tissue b r e a k d o w n h a d occurred this suggests a possible decline i n the respiratory d e m a n d of the older portions of the root.
Experiment I1 F a i l u r e to detect R O L a t the more distal portions of the longer roots a t room t e m p e r a t u r e raised the question of how such growth
M. T. Healy and W. Armstrong: Fig. 2
x ox ~ 2_ ~ -~ 0--
Fig. 2. R.O.L. at the apex of a pea root. i. with a jacket of 3% agar around all but
the apical region; 2. with the jacket completely removed; 3. as for 2 but after cooling to 3~ was sustained in the 3 % agar. Three possible reasons were considered: (a) t h a t the amount of internal oxygen was sufficient for growth only, leaving no excess for loss to the medium; (b) that the roots had grown anaerobically; (c) t h a t the agar had in effect reduced leakage from the root thus channelling sufficient oxygen to the apex to allow continued elongation. Since 3% agar substantially reduces oxygen diffusion, the last hypothesis seemed to merit further investigation: As before roots were grown in 3 % agar. At harvest, however, 1.5 cm of root apex only was exposed from the agar and this region was assayed for ROL. A further reading was then taken at the same apical position with all the remaining agar removed. Finally, the root was cooled, and a third apical assay taken. A typical example from the results is shown in Fig. 2. From this it appears t h a t the 3 % agar does have a " j a c k e t i n g " effect which reduces oxygen leakage, and thus allows a greater proportion of the oxygen entering the plant through the aerial parts to reach the root apex. I t seems likely t h a t the increase in the amount of available oxygen reaching the apex enabled growth to be prolonged. Fig. 2 also gives some indication of the comparative loss of available internal oxygen caused b y respiration and by leakage. Under the experimental conditions respiration is a greater sink than leakage. Under reducing conditions, i.e. with steeper gradients from root to soil, leakage will probably increase considerably in importance.
The Effectiveness of Internal Oxygen Transport
63%02 "// i 1 O0
BI 2 O0
Time (hours) Fig. 3. Root growth of pea under different aeration regimes. 1. o--o Roots transferred to aerated medium at A. and 63 % O3 medium at B. 2. ~---oRoots transferred to deoxygenated medium at A
Experiment I I I I t has been g e n e r a l l y a c c e p t e d t h a t m e s o p h y t i e p l a n t s in culture solutions require a e r a t i o n of t h e solution to ensure r o o t growth. H o w e v e r , t h e peas which grew in a g a r h a d no access to o x y g e n in t h e i r r o o t i n g m e d i u m . The effect of e x t e r n a l o x y g e n in a n a e r a t e d solution m a y therefore u n d e r certain circumstances be a k i n t o t h e " j a c k e t i n g " effect of t h e agar, i.e. to reduce o x y g e n loss from t h e r o o t r a t h e r t h a n s u p p l y o x y g e n for t h e r e s p i r a t o r y r e q u i r e m e n t s of t h e root. I n a n o t h e r e x p e r i m e n t t h e g r o w t h of roots in 3 % a g a r was r e c o r d e d for 3 days. T h e seedlings were t h e n dissected from t h e a g a r a n d d i v i d e d into two batches. The first were grown on w i t h roots in a e r a t e d 0.05% a g a r / w a t e r . A i r was b u b b l e d t h r o u g h t h e m e d i u m a t 12 h r i n t e r v a l s to m a i n t a i n aeration. T h e m e d i u m was u n d i s t u r b e d d u r i n g t h e i n t e r i m periods. The second were p l a c e d in d e o x y g e n a t e d 0.05% a g a r t h r o u g h which n i t r o g e n was p a s s e d a t similar intervals. The e q u i l i b r i u m O D R in t h e m e d i a were 5.6 • 10-Sg 0 2 c m - 2 m i n -1, a n d zero respectively.
M. T. Healy and W. Armstrong:
The growth rate in the aerated medium did not exceed t h a t in the non-aerated " j a c k e t " medium (3% agar), see Fig. 3, while boosting the oxygen regime with 63 % O 2 did not significantly alter the growth rate. Root growth in the oxygen-flee 0.05% agar was not arrested immediately. I t continued to match the rate of the air plants for c. 36 hr before it stopped. This continuation of growth might have been the result of internal oxygen supply, although the possibility of some anaerobic elongation cannot be overlooked. The effect produced b y aerating the 0.05% agar is consistent with the hypothesis previously stated t h a t the function of external oxygen is to mitigate oxygen loss from the root rather than to supply the root with respiratory oxygen. I n another experiment, peas were grown throughout with their roots in either the deoxygenated or aerated 0.05 % agar. Initially root growth occurred in the deoxygenated medium, but elongation of the main root stopped after reaching a length between 8 and 9 em. Aerated roots continued growing. Final Conclusions On the basis of the evidence given above, the following proposals are tentatively made in respect of the oxygen requirements of pea and other mesophytie species which do not readily form aerenchyma: (a) For root growth of an initial length (8-9 cm in the experimental plants), internally supplied oxygen will meet all respiratory requirements without the aid of a jacketing effect, other than t h a t afforded by a static oxygen-free culture solution in which diffusion gradients from root to medium will gradually become less and less steep. (b) After growth to a certain length or accumulation of respiratory tissue (including the formation of laterals), a more effective jacketing (e.g. a stiff agar or a static aerated medium) will be required if growth is to be prolonged. (c) Finally the accumulation of a larger respiratory sink in the more extensive root system m a y mean t h a t jacketing will become inadequate to ensure a supply of internal oxygen to the now effectively remote parts of the system. At this stage it will for the first time become necessary for the rooting medium to supply respiratory oxygen to the root system for growth to continue. One of us (M.T.H.)wishesto thank the Principal of Endsleigh College of Education, Hull, for the use of College facilities and her kindness in encouraging this work. We wish to thank l~r. R. Wheeler-Osman for preparing the figures.
The Effectiveness of Internal Oxygen Transport
References Armstrong, W.: Oxygen diffusion from the roots of some British bog plants. Nature (Lond.) 204, 801-802 (1964). Armstrong, W.: The use of polarography in the assay of oxygen diffusing from roots in anaerobic media. Physiol. Plant. 20, 540-553 (1967). Armstrong, W. : l~hizosphere oxidation in rice and other species: A mathematical model based on the oxygen flux component. Physiol. Plant. 23, 623-630 (1970). Cannon, W. A., Free, E. E. : Physiological features of roots with especial reference to the relation of roots to the aeration of the soil. Carnegie Inst. Wash. Publ. 368, 1-168 (1925). Crawford, R. M.M." The physiological basis of flooding tolerance. Ber. dtsch. Bot. 82, 111-114 (1969). Crawford, 1~. M. M., Tyler, P. D.: Organic acid metabolism in relation to flooding tolerance in roots. J. Ecol. 57, 235-244 (1969). Evans, N. T. S., Ebert, M. : Radioactive oxygen in the study of gas transport down the root of Vicia ]aba. J. exp. Bot. 11, 246-257 (1960). Greenwood, D. J. : Studies on the transport of oxygen through the stems and roots of vegetable seedlings. New Phytol. 66, 337-347 (1967a). Greenwood, D. J. : Studies on oxygen transport through mustard seedlings (Sinapsi8 alba. L.). New Phytol. 66, 597-606 (1967b). Heide, H. van der, l~aalte, M. H. van, Boer-Bolt, B.M. de: The effect of a low oxygen content of the medium on the roots of barley seedlings. Acta. Bot. Neerl. 12, 231-247 (1963). Hook, D. D., Brown, C. L., Kormanik, P. P. : Inductive flood tolerance in Swamp Tupelo (Nyssa sylvatica var. biflora (Walt.) Sarg). J. exp. Bot. 22, 78-89 (1971). Jensen, C. l~., Stolzy, L. H., Letey, J. : Tracer studies of oxygen diffusion through roots of barley, corn, and rice. Soil Sci. 103, 23-29 (1967). Leyton, L., l~ousseau, L. Z. : l~oot growth of tree seedlings in relation to aeration In: Physiology of forest trees (ed. by K. V. Thimann), chapt. 23, pp. 467. New York: Ronald Press 1957. Vartapetian, B. B. : Polarographic study of oxygen transport in plants. Fizioligia l~astenii 11, 774 (1964). Dr. W. Armstrong Department of Botany University of Hull Hull H U 6 71~X England