WATER-SENSITIVITY IN BARLEY I. RESPIRATION STUDIES AND THE INFLUENCE OF OXYGEN AVAILABILITY. By D. Crabb and B. H. Kirsop

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1 254 [J. Inst. Brew. WAER-SENSIIVIY IN BARLEY I. RESPIRAION SUDIES AND HE INLUENCE O OXYGEN AVAILABILIY By D. Crabb and B. H. Kirsop (Brewing Industry Research oundation, Nutfield, Redhill, Surrey) Received 12//i September, 1968 he respiration of water-sensitive and non water-sensitive barley has been investigated; it is shown that there is no significant difference in the rate of oxygen consumption by the two types of barley. he presence of a husk and a surface film of water are shown to reduce the oxygen uptake in both types of barley. he embryos of water-sensitive barleys germinate to a lesser degree than those of fully mature barleys at low oxygen tension. Water-sensitivity thus appears to be a reflection of a higher oxygen requirement for germination by the embryo. Introduction Since the recognition of as a factor responsible for the failure of viable barley grain to germinate4 and the description of methods for the measurement of its incidence,5 it has become widely recognized that slow and uneven germination of barley in malting may be due to the presence of a proportion of water-sensitive corns in the barley used. Such corns are unable to germinate, in an atmosphere of air, if their surface is covered with a film of water although they will do so in the absence of such water; this differentiates the condition, for practical purposes, from that of low germinative energy which is characterized by the failure of grain to grow however carefully the environment is regulated with regard to water.4 Water-sensitive barley can be induced to grow, in the presence of a coating of water, if germination is carried out in an atmosphere of oxygen,13-14'8 showing that it has a com paratively high requirement for oxygen if growth is to be initiated. he observation that removal of the husk and the pericarp enables water-sensitive barley to germinate in an atmosphere of air, in moisture conditions which inhibit the growth of untreated corns,1* again suggests that increased availability of oxygen allows growth to take place. It also seems likely that the presence of an external covering of moisture, which is necessary for the expression of water-sensitiyity, reduces oxygen availability by presenting an addi tional barrier to the diffusion of oxygen.8 In the course of work directed towards elucidating the underlying causes of watersensitivity it seemed desirable to establish to what degree the external coverings of the grain and an external water film reduce the penetration of oxygen to the embryo. It was also of interest to ascertain whether the degree of restriction exerted by these two factors was greater in the case of water-sensitive barleys than in those which were free from this condition. Manometric measurements were employed to give an estimate of the utiliza tion of oxygen in various circumstances. Results and Discussion When barley is immersed in aerated water the percentage of corns which germinate closely parallels the level of germination in the test (able I) and the level of growth in such circumstances can be used as an indication of when it is Relationship Between Water-sensitivity and Aerated Water A C D E G H JK L I aerated water (%) S

2 Vol. 75, 1969] CRABB AND KIRSOP: RESPIRAION O WAER-SENSIIVE BARLEY 255 Oxygen Uptake for Water-sensitive and Non Water-sensitive s During the 0-12 hr. Stage op Immersion in Aerated Water II germinative energy test (%) test (%) Mean O, uptake (fd./corn/br.) Husked Dehuskcd M N O B P Q present. he germination of barley immer sed in water in shaken manometric flasks was also dependent on the incidence of watersensitivity. It was thus possible to estimate the respira tory activity of corns under circumstances in which, when present, could prevent germination. When the up take of oxygen and evolution of carbon dioxide were assessed for a series of watersensitive and non water-sensitive barleys immersed in aerated water it was found that the mean rate of oxygen consumption measured over the first 12 hr. approximated to 1 /xl/corn/hr. (able II). his value 6 "o S. 5 r- 30 Hours of immersion ig. 1. Oxygen uptake by water-sensitive and non water-sensitive barley immersed in aerated water for 12 hr. 0 % Husked barley O O Dehusked barley (1) and (3) are water-sensitive barleys (2) is a non water-sensitive barley Results are expressed as a mean of two replicates for each treatment. is similar to ones reported earlier for other barley varieties.18 No significant difference could be found between the rates of oxygen consumption for water-sensitive and non water-sensitive barley (able II, ig. 1). Measurement of the rate of carbon dioxide evolution of two husked barleys, only one of which was water-sensitive, showed that this appreciably exceeded the rate of oxygen uptake; the respiratory quotients ( M, 2-8; O, 2-6) were thus substantially higher than unity, indicating that both barleys were respiring anaerobically to a significant degree in these circumstances (able II). When five of these barleys were examined in aerated water, after removal of the husk and pericarp by treatment with sulphuric acid,11 oxygen uptake occurred to a greater degree than in the untreated barleys (able II), indicating that these outer coverings were (ig. 1) a substantial barrier to the penetration of oxygen.8 he increase in the rate of oxygen uptake which followed the removal of husk and pericarp occurred in both types of barley. he increased availability of oxygen also resulted in the elimination of anaerobic respiration; the respiratory quo tient values were now close to unity for barleys M and O (able II). Both types of barley subsequently germinated when de husked. In more prolonged experiments, using husked barley, it was confirmed that no sub stantial difference in respiratory activity could be demonstrated between the barleys in which was present and those in which it was essentially absent. When germination eventually occurred, however, the increase in embryo activity was then reflected in an increased gas exchange. Similarly no difference could be detected in

256 CRABB AND KIRSOP: RESPIRAION O WAER-SENSIIVE BARLEY [J. Inst. Brew. Effects of a Surface ilm of Water on the Respiration of Water-sensitive and Non Watersensitive During the irst 12 hr.

3 256 CRABB AND KIRSOP: RESPIRAION O WAER-SENSIIVE BARLEY [J. Inst. Brew. Effects of a Surface ilm of Water on the Respiration of Water-sensitive and Non Watersensitive During the irst 12 hr. After Steeping III Water-sensitive barley, "0" Non vrntcr-sensltlvc barley "M" Bailey steeped for 48 Iir. Oxygen uptake (nl./corn/lir.) Co. evolution (nl./com/lir.) K.Q. Oxygon uptake <ixl./corn/lir.) Co. evolution (lil/corn/hr.) R.Q. Surface Him remaining Surface film removed by blotting the respiration rates of the two types of barley when they were dehusked prior to test (ig. 1) although the values were higher than in the case of husked grain. It is thus apparent that while the outer coverings of the grain represent a barrier to the penetration of oxygen to the embryo, the varying germina tion properties of the embryo cannot be attributed to differences in the degree of restriction exerted by the husk and pericarp. he influence of an external water film on respiratory activity was seen by examining corns previously steeped for 48 hr. and bear ing a naturally adhering film of water, in comparison with grain which had been similarly treated but from which this film had been removed by blotting. he un treated barleys utilized oxygen (able III) at the mean rate of 1-4 ^tl./corn/hr. whereas removal of external water resulted in a value of over 4 jd./corn/hr. Quite obviously super ficial water is a substantial barrier to the uptake of oxygen.9 he reduction in oxygen absorption resulting from this circumstance led to a degree of anaerobic respiration, as manifested by a high respiratory quotient (able III). Although the two barleys examined differed substantially in the inci dence of no significant differences could be detected in the degree to which oxygen uptake was diminished by the presence of external water. hus, although a high external oxygen tension is necessary if germination is to take place in water-sensitive barley when the moisture environment is unsuitable, it is apparent that such barleys do not differ from non-sensitive ones with regard to the inhibi tory effects either of water or of the seed coat on oxygen uptake. his in turn suggests that the enhancement of oxygen supply, which is necessary if germination is to take place, is indicative of a high oxygen require ment rather than of the existence in watersensitive barley of a greater external barrier to oxygen uptake than in corns which are free from this condition. On this view the availability of oxygen in the vicinity of the embryo is reduced to a low level by the combined effects of paleae, pericarp and external water. he embryo of a fully mature barley can germinate in these circum stances whereas that of a water-sensitive one cannot. As was expected, the rate of oxygen consumption by dehusked barleys immersed in water declined with oxygen tension (able IV). It is interesting to note that when oxygen tension was 4%, the oxygen uptake and CO2 production were similar to those of husked barley immersed in water at an oxygen Oxygen Consumption of Germinating at Different Oxygen ensions During the 0-12 hr. Stage of Immersion IV Oxygen tension and uptake of oxygen (/il./corn/hr.) Presence of husk 21% 10% 4% Husked barley O M Dehusked barley O M

Vol. 75, 1969] CRABB AND KIRSOP: RESPIRAION O WAER-SENSIIVE BARLEY 257 V VI Relationship Between Water-sensitivity and Comparison of Water-sensitivity with the the Germination ok Dehuskbd in Water

4 Vol. 75, 1969] CRABB AND KIRSOP: RESPIRAION O WAER-SENSIIVE BARLEY 257 V VI Relationship Between Water-sensitivity and Comparison of Water-sensitivity with the the Germination ok Dehuskbd in Water Growth of Dehusked in an Environment at a Low Oxygen ension of 4% Oxygen Germination after 72 hr. (%) Germination alter 72 hr. {%) Dchuskcd barley Water at 4% oxygen Husked barley Water-sensitivity test Husked barley Water-sensitivity test Dehusked barley Water-sensitivity test. Oxygen tension = 4% S R C U A D N V U c A W tension of 21%. his suggests that the oxygen tension in the immediate vicinity of the embryo, in the latter case, is of the order of 4% and that this low level of oxygenation is insufficient to permit germination to occur in the water-sensitive barley. If this is the case, dehusked barley should germinate to a degree related to the incidence of if placed in an atmos phere of low oxygen tension. When such corns are placed in aerated water they do, of course, germinate rapidly and completely. When, however, the oxygen content of the gas used for aeration was reduced to 4% the percentage which grew was reduced to the levels shown in able V. It is noteworthy that barleys varied in their level of germina tion at the lower oxygen tension in a similar way to their behaviour in the normal watersensitivity test, although the particular values for each barley in the two tests were not identical (able V). In further experiments the test was carried out with dehusked barley but with the atmosphere within and around the petri dishes at the low oxygen tension of 4% (able VI). Once again the barleys germinated to a varying degree and the order of decreasing germina tion was the same as that of husked watersensitive barley in the normal water-sensiti vity test. As in the previous case (able V) the actual percentage germination of a particular barley varied in the two sets of circumstances. However, the parallel between the ability of the dehusked grain to grow at low oxygen tension and the germination response of intact barley in the test, revealed in these trials, supports the view that is a reflection of a high oxygen requirement in the corn. hat this oxygen requirement is in fact a property of the embryo was shown by subjecting isolated embryos to low oxygen tension (2%) when VII Relationship Between Water-sensitivity and the Germination of Isolated Embryos at a Low Oxygen ension Germination of isolated embryos at 2% oxygen tension 2 ml. water 6 ml. water S D

$VIII Effect of Storage on the Oxygen Requirement by Dormant and Non-Dormant Date tested watersensitivity germinative energy test tot \ Germination (%) Oxygen 10% tension 4% Q Sept., 1000 Jan.$

5 258 CRABB AND KIRSOP: RESPIRAION O WAER-SENSIIVE BARLEY [J. Inst. Brew. VIII Effect of Storage on the Oxygen Requirement by Dormant and Non-Dormant Date tested watersensitivity germinative energy test tot \ Germination (%) Oxygen 10% tension 4% Q Sept., 1000 Jan., Sept., 1000 Jan., P Sept., 1900 Jan., those of non water-sensitive barleys germi nated to a much greater level than those of water-sensitive grain (able VII). It is known that the incidence of watersensitivity decreases to some degree, albeit slowly, during storage and it was of interest to ascertain the relationship between the germinative response of dehusked barley and oxygen tension before and after storage. he dehusked barleys were immersed in water through which gas containing varied propor tions of oxygen was passed. When first tested, four weeks after harvest, germination of three barleys examined was complete when the oxygen tension was 21%, but when this was reduced to 10% (able VIII) incomplete germination resulted in two of the three samples. When tested after 20 weeks storage at room temperature, the two barleys which had previously failed to grow at 10% oxygen tension now germinated fully, but failed to do so when the oxygen tension was reduced to 4%. his improvement in germinative be haviour was associated with the attainment of high germinative energy and a higher level of germination in the test. IX Effect of Oxygen on the Germinative Energy of B germinative energy Air Oxygen Quite clearly there is a diminished require ment for oxygen after storage. he failure to germinate completely in the first set of tests when oxygen tension was 10% is associated with the incidence of low germinative energy and it is possible that this condition is a manifestation of a still greater requirement for oxygen than is the case when watersensitivity alone is present. It is interesting to recall that occasionally barleys are so deeply dormant that they fail to germinate, when dehusked, in the aerobic environment of a germinative capacity test.10 urthermore, while low germinative energy is not com pletely removed, by carrying out tests in an atmosphere of oxygen, rather than air, germination is substantially improved (able IX). It is thus clearly possible that the amount of oxygen which penetrates the husk and pericarp even in the comparatively dry conditions of a germinative energy test is insufficient to meet the requirements of the embryo if this is deeply dormant. In this connection it is of interest to record that one treatment which removes low germinative energy, (heating grain in a sealed container at 40 C. for 72 hr.) does not alter the oxygen requirement of the embryo (able X). his treatment may well be improving oxygen availability, perhaps by reducing the barrier effect of the outer coverings of the grain, and particularly the activity of the oxidase systems known to be present in the pericarp12 and which it has been suggested are involved in the condition of low germinative energy. he present work shows that the condition of is a reflection of a high requirement for oxygen if the embryo of the grain is to germinate. he water condition

Vol. 75, 1009] CRABB AND KIRSOP: RESPIRAION O WAER-SENSIIVE BARLEY 259 X Effect of Heating for 66 hr. at 40 C, on the Germination Properties of! germinativc energy!

6 Vol. 75, 1009] CRABB AND KIRSOP: RESPIRAION O WAER-SENSIIVE BARLEY 259 X Effect of Heating for 66 hr. at 40 C, on the Germination Properties of! germinativc energy! 02 (33) P i 03 (30) Q 02 (85) i igures in parentheses refer to the unheatcd barley. 50 (10) 23 (13) 60 (44) Germination of dehusked grain at 10% oxygen tension (%) 63 (46) 62 (50) 78 (75) during germination and the external cover ings of the grain are of significance only in so far as they determine oxygen availability. Suggestions that bacterial contamination is responsible for the failure of water-sensitive barley to germinate1 >2 are not supported by these results, although bacterial activity might well exert an indirect effect by reducing oxygen supply to the embryo. It would seem that some oxidation reaction which readily takes place in the embryo of mature barley occurs with difficulty in the watersensitive embryo. he absence of any significant difference in oxygen utilization of water-sensitive and non water-sensitive grain supports earlier suggestions' that there are unlikely to be differences in the major oxygenutilizing system in the two types of grain and indicates that specific oxygen reactions, whether purely chemical or enzymic, may be involved. Earlier demonstrations' that par ticular compounds induce may well be a reflection of the inability of the embryo to overcome the effects of these materials in the relatively anaerobic circum stances of the test. If this view is correct, is a con sequence either of the presence of a specific inhibitory substance which can only be oxidized if the oxygen tension is high or alternatively of the absence of a suitable system to overcome the effects of such an inhibitor. Experimental Germination tests were carried out as described previously.8 he influence of oxygen tension on gaseous exchange or on germina tion was studied using oxygen or air or mixtures of these two gases with nitrogen. When grain was germinating in water the appropriate gas was passed continuously through the liquid to maintain a particular oxygen tension; the temperature of germina tion was 18 C. unless otherwise stated. All values for oxygen tension arc expressed as the percentage oxygen in the gas used. o measure gaseous exchange the direct method described by Umbreit, Burris & Stauffer16 was used. he temperature was 25 C. in all measurements of gas exchange. wenty barley seeds per Warburg flask were used in all cases and when the respiration of immersed grains was studied these were suspended in an antibiotic solution (3 ml.) containing streptomycin (1000 p.p.m.) nystatin (40 p.p.m.) and penicillin (58 p.p.m.); the germination properties of the immersed grains were not altered by the presence of these substances. Acknowledgement. he authors wish to thank Dr. A. H. Cook,.R.S., for his advice and encouragement during the course of this work. References 1. Blum, P. H., rcdcrickson, B. L., & Panos, G., Am. Brewer, Dec, 1060, Blum, P. H., & Gilbert, S. G., Proc. Amtr. Soc. Brew. Chem., Brown, R., Ann. Bot. Lond., 1943, 7, Essery, R. E., Kirsop, B. H., & Pollock, J. R. A., this Journal, C. Essery, R. E., Kirsop, B. H., & Pollock, J. R. A., this Journal, 1055, 25. Jansson, G.. Arhiv Kemi, 1959,14, 279. Jansson, G., Arhiv Kemi, 1962, Kirsop, B. H.. Proc. Irish Maltsters ech. Meeting, 1966, Ohmura,., & Howell, R. W.. PI. Physiol., Wash., 1960, 38, Pollock, J. R. A., Chem. & Ind., Pollock, J. R. A.. Essery, R. E., & Kirsop, B. H., this Journal. 1055, Pollock. J. R. A., & Kirsop, B. H., this Journal, Pollock, J. R. A., Kirsop, B. H.. & Essery. R. E., Proc. Eur. Brew. Conv Pollock. J. R. A., Kirsop. B. H.. & Essery, R. E. this Journal, 1955, Umbreit, W. W., Burris. R. H.. & Stauffer. J.., Manometric echniques & issue Metabolism. 2nd Edition, Minnesota: Burgess Publishing Co Urion, E., & Chapon, L., Proc. Eur. Brew. Conv., 1955, 172.

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