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Temperature effects on the response to sulphur of barley (Hordeum vulgare L.), peas (Pisum sativum L.)… Herath, Herath Mudiyanselage Walter 1970

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TEMPERATURE EFFECTS ON THE RESPONSE TO SULPHUR OF BARLEY (HORDEUM  VULGARE L.), PEAS (PISUM SATIVUM L.) AND RAPE (BRASSICA CAMPESTRIS L.) by HERATH MUDIYANSELAGE WALTER HERATH B.S.A., University of B r i t i s h Columbia, 1963 M.S.A., University of B r i t i s h Columbia, 1964 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Plant Science We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1970 In present ing th i s thes is in pa r t i a l f u l f i lmen t o f the requirements fo r an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee l y ava i l ab le for reference and study. I fu r ther agree tha permission for extensive copying o f th i s thes is for scho la r l y purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l i ca t ion of th is thes is fo r f inanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Depa rtment The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date f^l^-tl^ /7 i97V ABSTRACT The ef f e c t s of temperature and sulphur n u t r i t i o n on the growth, y i e l d and mineral composition (N, NO^-N, S and SO^-S) of Hordeum vulgare L. cv O l l i , Pisum sativum L. cv Dark Skin Perfection and Brassica campestris L. cv A r l o , were investigated i n controlled environments. The net C0 9 exchange rates and compensation points were also determined at two S leve l s (0 and 64 ppm) under various temperature regimes. When barley and rape plants were grown at 0 ppm S, deficiency symptoms developed i n about two weeks, whereas pea plants at the same l e v e l developed deficiency symptoms i n about three weeks. Plants at the lowest S l e v e l and the highest temperature took the shortest time to develop S deficiency symptoms. Fresh and dry weights, shoot length, number of nodes and number of f e r t i l e f r u i t increased with increasing S l e v e l s . Shoot growth i n a l l three species was more depressed by S deficiency than root growth. Optimum growing temperature regimes f o r barley and peas were found to be 24/16 at the vegetative stage and 18/10°C at the mature stage as evident from increased weights, maximum f r u i t set and mineral uptake. Optimum temperature for rape plants was 2 9/21°C at both stages of growth. i v Detrimental e f f e c t s of cotyledon or endosperm removal tended to mask the e f f e c t s of temperature and S l e v e l s . This method was thus found to be unsatisfactory fo r the study of S n u t r i t i o n i n plants. Higher mineral concentration was observed at the vegetative stage than at the mature stage i n peas and rape plants, while i n barley the mineral concentration remained constant at both stages of growth. With increase i n S supply there was an increase i n uptake of both t o t a l S and SO^-S. Uptake also increased with increasing temperatures. This increase was l a r g e l y due to "concentration e f f e c t s " . Hence the use of SO^-S l e v e l as a c r i t e r i o n f o r diagnosis of S def i c i e n c y may be unsatisfactory, unless plants are grown at optimum temperatures. S d e f i c i e n t plants had increased t o t a l N and NO^-N concentrations i n a l l three species. NOg-N concentration also increased with an increase i n temperature. The t o t a l N concentration did not increase appreciably with temperature. Consequently, at low S l e v e l (0 and 8 ppm) t o t a l N:total S r a t i o s (N:S) tended to i ^ r e a s e or decrease depending on low or high growing temperatures r e s p e c t i v e l y . These changes i n r a t i o s were independent of actual size of the plants. Furthermore the r a t i o s f o r a l l S le v e l s at the vegetative stage were lower than those at the mature stage. Therefore V both temperature and stage of growth are important factors to be considered i n inte r p r e t i n g S deficiency from N:S ra t i o s i n plants. The net C0 9 exchange rates were generally higher at 2 0 days than at 3 0 days. At 0 ppm S l e v e l and at high temperature, the decline i n net C0 9 exchange rate with age was greater. Maximum CO^ exchange rates were observed at the optimum growing temperatures f o r both S l e v e l s . Increasing the measuring temperature above the growing temperature caused no further stimulation i n C0 9 uptake, and at high temperatures there was a decrease i n uptake. When CC^ exchange rates were measured at two 5.5°C i n t e r v a l s above and below the growing temperatures the maximum rates were recorded at or below growing temperatures i n a l l the species at both S l e v e l s . The CC>2 compensation values were higher with lower S l e v e l i n the leaf tissue than at higher S l e v e l s . Increase i n growing temperatures also caused larger CC>2 compensation values than at lower temperatures. Negative correlations between C0 2 compensation point and l e a f tissue S l e v e l and pos i t i v e correlations between CC^ compensation point and temperature were observed i n barley and peas. v i TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 5 Ef f e c t of Temperature on Plant Growth 5 General Aspects of Temperature and Crop Growth 5 Temperature and Morphogenesis 9 Ef f e c t of Temperature on Some Biochemical Processes 10 Phys i o l o g i c a l Basis of Temperature Response 13 Response of Barley, Peas and Rape to Temperature 17 Barley 17 Peas 18 Rape 20 Mechanism of Ion Uptake 21 Transport of Minerals Within the Plant 24 Sulphur i n the N u t r i t i o n of Plants 26 Sulphur Deficiency and Relative Importance of Various Sources of Sulphur 26 Sulphate Sulphur i n S o i l s 29 Sulphur Oxidation i n S o i l s 31 Sulphur Deficiencies and Diagnostic Techniques for Determining S Deficiencies i n Crops and S o i l s 34 Sulphur Requirements f o r Various Crop Species 3 8 v i i Nitrogen-Sulphur Ratios i n Evaluating Sulphur Status of Various Species 39 Nitrogen and Sulphur Interactions i n Plants 4 0 Sulphur Metabolism 42 General Aspects 42 Role of Sulphur Compounds i n Structure and Their Function i n Metabolism 4 3 Carbon Dioxide Exchange 45 Photosynthesis 45 Respiration 47 Sulphur and Photosynthesis 50 Growth Stages and Photosynthesis 52 Adaptation to Temperature 5 3 MATERIALS AND METHODS 5 5 Plant Species 56 Growth Chambers 57 Cu l t u r a l Practices 58 Observations 59 Measurement of Compensation Point 61 Chemical Analysis 6 2 Preparation of Material f o r Chemical Analysis 6 2 Sulphate Sulphur Analysis Method 63 Total Sulphur 67 Total Nitrogen and Nitrate Nitrogen 6 8 Experimental Design and S t a t i s t i c a l Analysis 69 v i i i RESULTS 7 0 General Observations 70 E f f e c t of Sulphur N u t r i t i o n on Growth, Mineral Concentration and Total Uptake i n the Shoots at U Weeks (Vegetative Stage) as Influenced by Temperature 73 Growth 7 3 Nodes 78 Mineral Concentration and Total Uptake 78 Total Sulphur 7 8 Sulphate Sulphur Concentration and Total Uptake 80 Nitrogen Concentration and Total Uptake 82 Nitrogen/Sulphur Ratio 83 E f f e c t of Temperature and Sulphur N u t r i t i o n on the Growth, Mineral Concentration and Total Mineral Uptake i n Barley, Peas and Rape Plants at Mature Stage 83 Growth 83 Nodes 87 Mineral Concentration and Total Uptake 87 Total Sulphur Concentration 87 Total Uptake of Sulphur 89 Sulphate Sulphur Concentration 89 Total Uptake Sulphate Sulphur 91 Total Nitrogen Concentration 91 Total Nitrogen Uptake 92 ix Nitrate Nitrogen Concentration 9 2 Total Nitrate Nitrogen Uptake 9 3 N:S Ratio 9 3 E f f e c t of Temperature and Sulphur N u t r i t i o n on the Y i e l d and Mineral Concentration i n F r u i t s of Barley, Peas and Rape 93 Sulphate Sulphur Concentration 9 5 Total Uptake of Sulphate Sulphur 9 5 Nitrogen Concentration 9 6 Total Uptake of Nitrogen 96 F r u i t Weight as Percent of Total Dry Weight 9 7 E f f e c t of the Removal of Cotyledons or Endosperm on Sulphur Responses of Barley, Peas and Rape 97 Growth 9 7 Nodes 99 E f f e c t of Temperature and Sulphur N u t r i t i o n on Net C O 2 Exchange Rates of Barley, Peas and Rape at 20 and 30 Days 99 E f f e c t of Temperature and Sulphur N u t r i t i o n on C O 2 Compensation Points i n Barley, Peas and Rape 109 DISCUSSION . 116 Development of Sulphur Deficiency Symptoms i n the Three Species 116 Growth Responses to Sulphur N u t r i t i o n 118 E f f e c t of Removal of Cotyledons or Endosperm on Sulphur Responses 122 X Mineral Concentration and Uptake 12 3 Photosynthesis 134 C0 9 Compensation Point 139 SUMMARY AND CONCLUSIONS 144 BIBLIOGRAPHY 150 APPENDICES 1. Diagram Showing Random Arrangement i n Each of Three Rows i n the Growth Chamber 17 0 2. An Example of a Combined Analysis of Variance f o r Main E f f e c t s 171 x i LIST OF TABLES Page Table 1. F values and the sig n i f i c a n c e s of main eff e c t s and interactions for each measurement at 4 weeks (vegetative Stage). 74 Table 2. E f f e c t of temperature and sulphur n u t r i t i o n on the growth of barley, peas and rape plants at 4 weeks (vegetative stage). Experiment 1 75 Table 3. Growth c h a r a c t e r i s t i c s and y i e l d factors i n barley, peas and rape as influenced by temperature and sulphur n u t r i t i o n at 4 weeks (vegetative stage). Experiment 4 77 Table 4. E f f e c t of temperature and sulphur n u t r i t i o n on the mineral concentration and t o t a l uptake per plant shoot, i n barley, peas and rape at 4 weeks (vegetative stage). Experiment 4 79 Table 5. E f f e c t of temperature and sulphur n u t r i t i o n on the concentration and t o t a l uptake of sulphate sulphur and nitrogen i n the shoots of barley, peas and rape at 4 weeks (vegetative stage). Experiment 2 81 Table 6. E f f e c t of temperature and sulphur n u t r i t i o n on the growth of barley, peas and rape at mature stage. Experiment 5 84 Table 7. E f f e c t of temperature and sulphur nutr-i t i o n on the growth of barley, peas and rape at mature stage. Experiment 3 85 Table 8. E f f e c t of temperature and sulphur n u t r i t i o n on the concentration of sulphur and nitrogen i n the shoots of barley, peas and rape at maturity. Experiment 5 8 8 x i i Table 9. E f f e c t of temperature and sulphur n u t r i t i o n on the concentration of sulphate sulphur and t o t a l nitrogen i n the shoot of barley, peas and rape at mature stage. Experiment 3 Table 10. E f f e c t of temperature and sulphur n u t r i t i o n on the y i e l d , sulphate sulphur and nitrogen concentration i n the f r u i t s of barley, peas and rape. Experiment 5 Table 11. E f f e c t of excision of cotyledons or endosperm and sulphur n u t r i t i o n on the growth and y i e l d factors of barley, peas and rape at 4 weeks (vegetative stage). Experiment 6 Table 12. E f f e c t of excision of cotyledons and sulphur n u t r i t i o n on the growth of nodes of barley, peas and rape at 4 weeks (vegetative stage). Experiment 6 90 94 98 100 Table 13. F values and the si g n i f i c a n c e s of main e f f e c t s and interactions of net COo exchange rate for barley, peas and rape. Experiment 7 101 Table 14. E f f e c t of temperature and sulphur n u t r i t i o n on net C O 2 exchange rates of barley at two growth stages. Experiment 7. Ef f e c t of temperature and sulphur n u t r i t i o n on net C O 2 exchange rates of peas,at two growth stages. E f f e c t of temperature and sulphur n u t r i t i o n on net C O 2 exchange rates of rape plants at two growth stages. Experiment 7 2 Table 17. Ratios of mgCO^/hr/dm exchanged i n plants grown with 64 ppm sulphur and 0 ppm sulphur. Measurements compared at the growing temperatures and at 5.5°C higher. Experiment 7 Table 15. Table 16. 103 105 107 110 X l l l Table 18. F values and s i g n i f i c a n c e s of main ef f e c t s of temperature and sulphur l e v e l and t h e i r i n t e r a c t i o n . Experiment 8 Table 19. Main e f f e c t s of temperature on C O 2 compensation and sulphur l e v e l i n l e a f t i s s u e . Experiment 8 Table 20. E f f e c t of temperature and sulphur n u t r i t i o n on the C O 2 compensation point i n barley, peas and rape. Experiment 8 Table 21. R squared values from regression analysis ( C O 2 being dependent v a r i a b l e ) . Experiment 8 111 112 114 115 xiv LIST OF FIGURES Page Fi g . 1. Apparatus for the reduction of sulphate (Gustafsson, 1960b) 65 F i g . 2. The 6 unit apparatus used f o r microestimation of sulphur 66 F i g . 3a and b. E f f e c t of sulphur l e v e l s (0, 8 and 64 ppm) on growth of barley and peas at 18/10°C (day/night). Ten weeks from sowing. 71 F i g . 3c. E f f e c t of sulphur l e v e l s (0, 8 and 64 ppm) on the growth of rape at 18/10°C (day/night). Ten weeks from sowing. 72 X V ACKNOWLEDGEMENTS I wish to thank Dr. Douglas P. Ormrod, formerly Professor, Department of Plant Science, University of B r i t i s h Columbia and presently Professor and Chairman, Department of H o r t i c u l t u r a l Science, University of Guelph, Ontario, under whose supervision t h i s study was undertaken, for his constant i n t e r e s t , encouragement and advice during the research, and for his guidance i n the preparation of t h i s t h e s i s . Deep appreciation i s extended, for i n d i v i d u a l and c o l l e c t i v e advice, and for the review of t h i s t h e s i s , to the members of my graduate committee: Dr. V.C. Brink, Department of Plant Science Dr. G.W. Eaton, Department of Plant Science Dr. W.D. K i t t s , Chairman, Department of Animal Science Dr. L.E. Lowe, Department of S o i l Science Dr. E.B. Tregunna, Department of Biology and Botany I am grat e f u l to Dr. G.W. Eaton for his valuable assistance i n the computer programming of the data. I thank Mr. Ilmars Derics, technician, f o r help with laboratory equipment and Miss Heather Barr and Mr. Ashley Herath f o r t h e i r t echnical assistance i n carrying out t h i s project. Thanks are also expressed to my colleagues Messrs. Wayne Fleming and Frank Eady fo r t h e i r help given from time to time and to Mrs. Connie Crossling and Miss Retha Holzman fo r accurate typing of t h i s t h e s i s . x v i Grateful acknowledgement i s given for f i n a n c i a l assistance received from the Sulphur I n s t i t u t e , Washington, D.C. and to the University of B r i t i s h Columbia f o r a Graduate Fellowship. INTRODUCTION From the time of L i e b i g sulphur has been described as one of the e s s e n t i a l elements for plant growth (Coleman, 1966). Its importance was not recognized u n t i l recent times when crop y i e l d s were beginning to decline i n many parts of the world. S o i l d e f i c i e n c i e s of S have been reported with increasing frequency throughout the world, e s p e c i a l l y i n the United States, Canada, B r a z i l , A u s t r a l i a , New Zealand and several countries i n A f r i c a and Asia. In Europe S d e f i c i e n c i e s have been reported i n France, West Germany, Norway, Sweden and Spain. S d e f i c i e n c i e s occur p r i m a r i l y due to increased use of S free f e r t i l i z e r s ; decreased use of S containing fungicides and i n s e c t i c i d e s ; and the production of higher y i e l d i n g crops which are placing a greater demand upon the S supplying power of the s o i l . S i s an e s s e n t i a l element i n the growth of plants, as i t i s a constituent of methionine and cysteine, two amino acids commonly found i n plants. Methionine i s e s s e n t i a l i n animal n u t r i t i o n and cysteine, although not e s s e n t i a l , may complement methionine when the l a t t e r i s present i n suboptimal amounts. Furthermore two vitamins, thiamine and b i o t i n , contain S. It also occurs i n several other compounds such as a c e t y l coenzyme A and mustard o i l glycosides. 2 The a v a i l a b i l i t y of S to plants depends on several f a c t o r s . Usually i f the s o i l has adequate amounts of S, plants are able to grow normally, providing the environmental factors such as temperature are not l i m i t i n g . I f , on the other hand, the S l e v e l i n a s o i l i s below the optimum plant require-ment , growth may be retarded and growing temperature may have an enhanced e f f e c t , such as on the earliness and the i n t e n s i t y of the S deficiency symptoms. In other words, the requirements of S f o r a p a r t i c u l a r species may be greater or less depending on the p r e v a i l i n g temperatures. On t h i s basis i t should be possible to predict the S requirements of crops f o r a p a r t i c u l a r season. Barley and peas are "cool season" crops and can therefore be successfully grown only during periods when temperatures are r e l a t i v e l y low. Their temperature require-ments may be d i f f e r e n t at d i f f e r e n t stages of growth. Rape plants i n contrast have a wide range of a d a p t a b i l i t y and may grow well at both high and low temperatures throughout the season. Many experiments have been c a r r i e d out to study the e f f e c t of S on crop y i e l d s , shoot weights and root weights, yet the responses of various species with respect to t h e i r morphological development such as shoot length, root lengths, number of nodes, have r a r e l y i f ever been included. Further-more the ad a p t a b i l i t y of a species to a p a r t i c u l a r temperature 3 may depend on the l e v e l of S a v a i l a b l e to the plant. Its p h y s i o l o g i c a l functions may change as do morphological and anatomical structures with changes i n external growing conditions p a r t i c u l a r l y temperature. The adaptive f l e x i b i l i t y of the species could be l i m i t e d under S d e f i c i e n t conditions. Very l i t t l e evidence i s a v a i l a b l e on the e f f e c t s of temperature on S uptake by plants. A notable contribution was that of McKell and Wilson (1963) who worked with rose and subterranean clover. The e f f e c t of temperature and S l e v e l s on net CC^ exchange rates, has not been investigated, although i t was evident that plants grown under low S l e v e l s have reduced photosynthetic rates (Thomas et al_. , 1943), mainly due to lack of chlorophyll i n the shoots of S d e f i c i e n t plants (Ergle, 1932). The s p e c i f i c objectives of the present studies were to determine the e f f e c t s of temperature on: 1. Pattern and rate of development of S deficiency symptoms i n barley, peas and rape, 2. Growth c h a r a c t e r i s t i c s and y i e l d factors at d i f f e r e n t S le v e l s , 3. S uptake and d i s t r i b u t i o n i n the d i f f e r e n t parts of the plant at vegetative and mature stages of growth. 4 4. The uptake and d i s t r i b u t i o n of t o t a l N and NOg-N at d i f f e r e n t S l e v e l s . Also to determine: 1. The optimum temperatures at vegetative and mature stages for e f f i c i e n t production of barley, peas and rape, and u t i l i z a t i o n of plant nutrients 2. The e f f e c t s of removal of cotyledons or endosperm on the response of the three species to S at three temperature regimes 3. The e f f e c t of temperature and S l e v e l s on the t o t a l N: t o t a l S r a t i o s at vegetative and mature stages of growth 4. The e f f e c t s of S on the CC^ exchange rates i n the three species at various growing and measuring temperatures at 2 0 and 3 0 days from sowing 5. Changes i n the CC^ compensation points i n the three species, due to changes i n the growing temperatures and S l e v e l s of l e a f t i s s u e . 5 LITERATURE REVIEW Ef f e c t of Temperature on Plant Growth  General Aspects of Temperature and Crop Growth Thermal energy or heat i s one of the most important environmental factors that affects the growth and development of l i v i n g organisms. A l l organisms are continually subjected to some degree of heat, and i t i s the magnitude of t h i s that i s important to the organism. D e f i n i t i v e information on the e f f e c t of heat on l i v i n g organisms came only a f t e r the construction and development of suitable instruments for making accurate measurements of temperature (Rose, 1967). Heat i s a form of k i n e t i c energy which can be transformed into other kinds of energy or can be transmitted from a r e l a t i v e l y warm body to a colder one. This t r a n s f e r of heat i s constantly going on, and the d i r e c t i o n and rates of trans f e r comprise one of the most important aspects of the organic environment. Transfer takes place by r a d i a t i o n , convection and conduction (Daubenmire, 19 59). The r o l e of temperature i n crop production i s apparent from the s p e c i f i c i t y of the c u l t i v a t e d species grown i n the subarctic, temperate, and t r o p i c a l regions. The wide range of cu l t i v a t e d species found among these regions and t h e i r d e f i n i t e seasonal r e l a t i o n s are brought about by a r e l a t i v e l y narrow temperature range. Within t h i s range b i o l o g i c a l a c t i v i t i e s are e s s e n t i a l l y restricted, to temperatures between 0 and 60°C. 6 This range i s l i m i t e d on the lower side by the freezing point of water and on the upper side by the heat of denaturation of proteins (Leopold, 1964). Crop production research on plant-temperature r e l a t i o n s f a l l s into three general categories. F i r s t i s the determination of the optimum temperature regime f o r the various crop species. Second i s the maximum temperature endured by the crop species without eith e r reduced dry matter accumulation or death of the plant. Third i s the lowest temperature tolerated by the crop species, which f o r some species may be much below freezing and f o r others well above freezing (McCloud et a l . , 1964). The provision of better c o n t r o l l e d environment f a c i l i t i e s i n recent years has provided a great deal of information of t h i s sort (Went, 1953). However, attempts to e s t a b l i s h f i x e d c a r dinal temperatures, p a r t i c u l a r l y optimum temperatures, have been inconclusive. The ph y s i o l o g i c a l complexity of the plant as an organism may preclude the d e f i n i t i o n of these ca r d i n a l points, because d i f f e r e n t p h y s i o l o g i c a l processes within the plant may have d i f f e r e n t temperature c o e f f i c i e n t s . Although the d e f i n i t i o n of these cardinal temperature points may remain empirical, there i s presumably an optimum, maximum and minimum for each crop variety grown under a given set of environmental conditions. Different functions of the same plant may have d i f f e r e n t c a r d i n a l temperatures. In many plants, i f not most, the optimal temperature f o r photosynthesis i s d i s t i n c t l y lower 7 than the optimum f o r r e s p i r a t i o n . Within species the c a r d i n a l temperatures vary f o r the three major phases of development, namely germination, vegetative growth, and reproduction (Daubenmire, 19 59). Most pertinent to crop production i s the optimum temperature regime. Some crops l i k e corn, sorghum and soya bean need much warmer temperatures f o r maximum y i e l d s than other crops l i k e oats, barley, peas and potatoes. Optimum temperatures represent a compromise between two opposing p h y s i o l o g i c a l pro-cesses. As temperature i s increased beyond a p a r t i c u l a r temperature the rate of photosynthesis decreases, but the rate of r e s p i r a t i o n increases (Ormrod and Bunter, 1961). Thus, the r e l a t i v e growth rate of plants grown at high temperature decreases. With time, at high temperature both net a s s i m i l a t i o n rate and l e a f area per unit plant weight decline. This i l l u s -trates another d i f f i c u l t y involved i n attempting to e s t a b l i s h the c a r d i nal temperatures. The reduced growth rate of plants below optimum temperature i s generally considered a r e s u l t of decreased rate of chemical reactions. However, no reasonable explanation has been advanced f o r the observed range of growth of d i f f e r e n t crop species at temperatures near 10°C. For example, corn and tomatoes cannot survive such conditions whereas the cool season grasses, some st r a i n s of a l f a l f a and small grains are able to maintain substantial net a s s i m i l a t i o n rates (McCloud et a l . , 1964). 8 Went (1948) and Mitchel and Lucanus (1960) observed that low day temperatures gave a much greater r e l a t i v e reduction i n growth than did lower night temperatures. Presumably t h i s i s a r e f l e c t i o n of lower photosynthetic rates as a r e s u l t of low day temperatures. Diurnal changes i n temperature, normally associated with the l i g h t and dark period,produce pronounced e f f e c t s on plant growth and development. Thermoperiodic e f f e c t s have been extensively investigated and Went (1953, 1957) reviewed the l i t e r a t u r e on t h i s subject. Many plants are adapted to f l u c t u -ating temperatures and develop optimally under such conditions. In the absence of such conditions growth may be abnormal as i n tomato plants kept under constant temperature and continuous i l l u m i n a t i o n (Went, 1948; and Hillman, 1956). Growth of these plants can be restored to normal by resuming diurnal fluctuations of either l i g h t or temperature. Peas grown under conditions of constant l i g h t and temperature are more variable i n phenotype than g e n e t i c a l l y i d e n t i c a l peas grown i n a f l u c t u a t i n g environment (Went, 1957). Highkin (1958) grew peas at a constant temperature of 10°C and noted a growth i n h i b i t i o n of approximately 20 per cent i n comparison with what i t would have been had diurnal f l u x of temperature been experienced. This i n h i b i t e d state was transmitted to subsequent generations, becoming cumulatively worse when grown under constant temperature. It took three generations of growth under a l t e r n a t i n g temperatures to restore the f u l l natural growth. Other p h y s i o l o g i c a l processes 9 are also affected by such diurnal temperature changes. Robertson et a l . (19 62) reported that diurnal temperatures have a pronounced e f f e c t on the carbohydrate composition of developing pea seeds. At low night temperatures the conversion of sugars to starch i n developing seeds was delayed, whereas at high temperatures the sugars were r a p i d l y converted. Temperature and Morphogenesis Temperature can a l t e r the morphology of plant organs or even whole plants and change the developmental patterns. Although patterns are usually f i x e d they may be reversed by a return to the o r i g i n a l temperature. These temperature influences d i f f e r from the long-lasting and often permanent inductive e f f e c t s such as v e r n a l i z a t i o n and dormancy i n that they respond to the immediate environment. This morphological f l e x i b i l i t y enables plants to adapt successfully to f l u c t u a t i n g environments. The capacity to change applies only to morphogenetic centres such as the growing points which are capable of continued growth. These centres respond to temperature d i r e c t l y by modifying the rate or pattern of l e a f primodia production or by developing f l o r a l organs. During the i n i t i a t i o n and expansion of leaves, temperature may profoundly influence t h e i r eventual size and shape presumably through some control exercised at the time of c e l l d i v i s i o n and c e l l expansion. Friend (1965) has shown that an increase i n temperature over the range 10 to 30°C 10 r e s u l t s i n narrower and thinner cereal leaves. This e f f e c t of temperature on l e a f length and width was brought about primarily by an increase i n c e l l length. The number of c e l l s along the lamina showed l i t t l e v a r i a t i o n . S t a n f i e l d et a l . (1966) reported increased numbers of nodes before flowering i n peas when grown at higher temperatures. Although a good deal i s known at the d e s c r i p t i v e l e v e l with regard to the e f f e c t of environmental factors such as temperature on development, v i r t u a l l y nothing i s known of the mechanisms whereby these factors modify the phenotype. Possible mechanisms include e f f e c t s on metabolic rates c o n t r o l l i n g the a v a i l a b i l i t y of substrate, p a r t i c u l a r l y carbohydrate, for growth and changes i n the regulation of endogenous le v e l s of hormones i n the tissue (Booth e_t al_. , 1962 ; Bollard and Wildman, 1964; and Hurd, 1964). Ultimately at the l e v e l of the d i f f e r e n t i a t i n g organ, the v i s i b l e a l t e r a t i o n s i n form are brought about by changes i n the rate or plane of c e l l d i v i s i o n . E f f e c t of Temperature on Some Biochemical Processes Because heat increases the k i n e t i c a c t i v i t y of the molecules, the higher the temperature the more rapid i s the rate of chemical reaction and consequently the rate of the physiologic processes. The r a t i o of a rate of reaction at a given temper-ature to i t s rate at a temperature 10°C lower i s c a l l e d the temperature c o e f f i c i e n t and i s designated by the symbol Q^Q (Daubenmire, 1959). I t i s r e l a t i v e l y low (1.2 to 1.4) f o r 11 physical reactions such as d i f f u s i o n , and f o r photodriven reactions. For enzymatic reactions the Q^g values may be markedly higher, ranging from 1.3 to 5, but commonly they range around 2. Unlike a physical process, as heat energy i s applied to a reactant i n an enzymatic reaction, the increased disordered energy leads to much larger increases i n the frequency at which the reactants reach the energy of a c t i v a t i o n needed fo r enzymatic c a t a l y s i s and the reaction rate i s increased more nearly exponentially (Leopold, 1964). Temperature changes can a l t e r the s o l u b i l i t y of and which are the two basic ingredients f o r metabolic a c t i v i t i e s . At r e l a t i v e l y low temperatures large amounts of and increased amounts of 0 2 can be held i n the sap of plants. This i n turn w i l l have an e f f e c t on enzymatic processes which involve exchange of these two gases (Leopold, 1964). Went (1953) and several other workers have attributed the biochemical differences i n many plants to temperature and other c l i m a t i c f a c t o r s . For example, i t has been found that the content of aromatic compounds i n tea plants varies with a l t i t u d e . When the a l t i t u d e i s higher the tea i s more aromatic. When growth slows, at usually above optimum temperatures, i t can be restored by addition of a single compound. I f the growing temperature i s r a i s e d a l i t t l e more, a further substance i s required and these requirements become progressively more numerous with increased temperature. Most of the high temperature-induced growth d e f i c i e n c i e s can be 12 repaired with the feeding of s p e c i f i c substances (Langridge and G r i f f i n g , 1959). Bonner (1957) referred to these growth d e f i c i e n c i e s as temperature l e s i o n s . Some of the organic compounds required at high temperature are thiamine, methionine, b i o t i n , cysteine and pantothenic acid. At low temperatures, methionine and thiamine are required among a host of other organic compounds. Schroeder (1963), on the other hand, suggests a mechanism whereby conditioning of a tissue by thermal shock can i n a c t i v a t e heat s e n s i t i v e enzyme systems, allowing the more heat l a b i l e enzymes to b u i l d up products or by-products which can then be made r e a d i l y a v a i l a b l e f o r further reactions at high temperatures. The r e l a t i o n of SH groups to f r o s t resistance has been shown i n many ways by L e v i t t 'et al_. (1961, 1962), L e v i t t (1962), and Schmutz et al_. (1961). When cabbage or saxifrages were frozen and t h e i r SH content determined before and a f t e r freezing no appreciable change was observed i f the freeze was non-i n j u r i o u s , but there was a marked decrease i n SH i f the freeze k i l l e d the plant. During the hardening process the SH content of plants i s known to increase, e s p e c i a l l y during the early stages. This has been demonstrated by Schmutz et al_. (1961) i n a series of wheat v a r i e t i e s d i f f e r i n g i n hardiness. They observed a high c o r r e l a t i o n between hardiness and SH content. If the plant i s f r o s t r e s i s t a n t i t can be frozen without i n j u r y 13 because i t s SH groups r e s i s t oxidation to SS. The r o l e that freezing plays i n the process i s to dehydrate the protoplasm and to bring the sulphydryl groups of adjacent proteins close enough together to permit them to react with each other. Due to the formation of intermolecular SS bonds, adjacent protein molecules may become joined together on freezing and t h i s may lead to unfolding of the molecules on rehydration during thawing. Links may also occur due to SH.^=±SS interchange and therefore do not require oxidation of the SH. This interchange i s due to a chain reaction which may be i n i t i a t e d by a small molecule SH substance. This could be the reason why some a c t i v e l y growing plants with high SH group contents cannot survive freezing. The high SH may be p a r t l y due to glutathione (GSH) which can act as a primer for such chain reactions between proteins. During the hardening process, the GSH o x i d i z i n g a c t i v i t y of the tissues increases as the GSH i s oxidized to GSSG. In t h i s form i t can no longer act as a primer, and the tiss u e can be frozen without i n i t i a t i o n of t h i s chain reaction ( L e v i t t , 1963). Physiological Basis of Temperature Response We can d i s t i n g u i s h between the d i r e c t temperature ef f e c t s on p h y s i o l o g i c a l p a r t i a l processes and the d i r e c t temperature e f f e c t s on the organism as a whole. C e l l growth, e s p e c i a l l y c e l l elongation, has a high Q^Q which indicates that t h i s i s a chemically rather than p h y s i c a l l y c o n t r o l l e d phenomenon 14 (Chao and Loomis, 1947). There are many studies i n which d i f f e r e n t i a l e f f e c t s of temperature on r e s p i r a t i o n and photosynthesis were found. For example, the work of Decker (1944) and Wager (1941) showed that at lower temperatures the r a t i o of photosynthesis to r e s p i r a t i o n i s over 10, and at higher temperatures, r e s p i r a t i o n i s r e l a t i v e l y increased and thus there i s a lower P/R r a t i o . One fa c t o r which complicates any analysis of the e f f e c t of temperature on photosynthesis is' the p o s s i b i l i t y of i n t e r n a l l e a f temperatures being either higher or lower than the ambient a i r temperature (Langridge and McWilliam, 1967). The needles of c o n i f e r s , f o r example, on a sunny day i n winter may have an i n t e r n a l temperature 2 0°C higher than the a i r temp-erature. Increases of such a magnitude would have a profound influence on the photosynthetic a c t i v i t y of evergreens i n winter, as the temperature difference i s usually greatest when a i r temperatures are low. Salisbury and Spomer (1964) observed l e a f temperatures well above the ambient temperature i n some alpine plants and cooling of leaves at high a i r temperatures. The adaptive s i g n i f i c a n c e of such modifications of i n t e r n a l temperatures of leaves has been demonstrated by many workers. .Changes i n the rate of photosynthesis with temperature are complex. In general under normal concentrations of C0 2 (about 3 00 ppm) and saturating l i g h t i n t e n s i t i e s , the increase i n the rate of f i x a t i o n of C0 9 with increasing temperature i s greatest at temperatures just above 0°C. According to Leopold 1 5 ( 1 9 6 4 ) the r e l a t i v e l y high Q^Q values reported f o r photosynthesis at low temperatures r e f l e c t the l i m i t a t i o n s imposed by bio-chemical processes. At high temperatures photosynthesis decreases ra p i d l y to zero due to thermal impairment of the photosynthetic apparatus. The rapid decline i n apparent photosynthesis with increasing temperature above the optimum i s also caused by the increase i n r e s p i r a t i o n (Decker, 1 9 4 4 ) . According to Geronimo and Beevers ( 1 9 6 4 ) temperature also influences the age dependent decline i n r e s p i r a t o r y mitochondrial a c t i v i t y i n plant leaves. High temperatures accelerate, and low temperatures decelerate t h i s decline, which occurs normally even at optimum temperatures. Friend ( 1 9 6 5 ) proposed that the decreased growth rate with increasing temperature above the optimum i s mainly caused by increased r e s p i r a t o r y losses during the dark period. He also found that the r a t i o of photosynthesis to r e s p i r a t i o n i s inversely r e l a t e d to temperature. The a v a i l a b l e evidence supports the theory that temperature influences the t r a n s l o c a t i o n of organic compounds within the plant, and that increase i n temperature causes an increase i n the rate of translocation with an optimum between 2 5 and 3 0°C and a Q^Q value greater than unity (Hewitt and C u r t i s , 1 9 4 8 ; Swanson and Bohning, 1 9 5 1 ; Swanson, 1 9 5 9 ; and Whittle, 1 9 6 4 ) . Translocation i s a complex phenomenon and i s made up of a series of sequential processes which are influenced to a greater or l e s s e r extent by temperature. These include the a v a i l a b i l i t y of materials i n the leaves and t h e i r movement into the translocating pathway from the as s i m i l a t i n g organ, both of which processes depend on metabolic a c t i v i t y . Also included i s the r a d i a l translocation of materials from the conducting elements into the surrounding tissue and f i n a l l y t h e i r removal at s i t e s of high metabolic a c t i v i t y . For example, i n roots, which are important s i t e s of metabolic a c t i v i t y , the temperature, i f lowered, would decrease the rates. Burr et a l . (1958) and Hartt (1965) p a r t i c u l a r l y observed t h i s i n the accumulation of sugar i n sugar cane. C h i l l i n g of roots causes a greater decrease i n the movement of organic compounds than c h i l l i n g shoots, suggesting that the main e f f e c t of temperature i s on the metabolism of the roots. The majority of recent studies support the view that the tr a n s l o c a t i o n of sugars i s retarded when the plant i s subjected to low temperatures. Many hypotheses have been put forward such as increase i n v i s c o s i t y and e f f e c t on r e s p i r a t o r y reactions as stated by Esau et a l . (1957). Although less s p e c i f i c , the most important i s the e f f e c t of low temperature on the centres of growth which constitute the main s i t e s for metabolism of substances moving i n the plant. The importance of such s i t e s of metabolic a c t i v i t y i n determining the v e l o c i t y of movement i s well established (Nelson, 1962; and Thrower, 1965) and i t i s becoming cle a r that the regulation of t h e i r a c t i v i t y by temperature i s a major factor c o n t r o l l i n g the pattern of translocation over the enti r e plant. The av a i l a b l e information 17 suggests that the movement of materials along the trans l o c a t i n g pathway i n the conducting tissue of the p e t i o l e or stem i s la r g e l y independent of temperature, but that the movement into and out of the pathway i s temperature dependent, mainly because i t i s linked to metabolic processes. Response of Barley, Peas and Rape to Temperature Barley Although barley i s not p a r t i c u l a r l y winter-hardy, most v a r i e t i e s grow best i n a coo l , moist climate. Some of the spring types of barley mature i n as l i t t l e as 60 days while winter types require as much as three times that long (Wolfe and Kipps, 19 59). Winter v a r i e t i e s are sown i n the cooler temperatures of the f a l l and harvested i n 'the la t e spring and early summer, before high humidity and high temperature set i n (Weaver, 1950). Generally for spring barley c u l t i v a t i o n a summer temperature around 21°C i s found to be conducive to s a t i s f a c t o r y growth. Ormrod e_t a l . (19 68) reported a maximum CC^ exchange rate between 16 and 20°C from t h e i r study of the e f f e c t of temperature on net C0 2 exchange rates of several barley v a r i e t i e s . Faris (1968) studied the germination time i n barley as affected by temperature and observed that at the low temperature of 4°C germination took 14 to 20 days more than at 2 8°C. Scheibe and Elberman (1968) reported that the amount of t i l l e r i n g i n barley and oats increased with increase i n temperature from 6 to 20°C. 18 Power et a l . (1963) studied s o i l temperature and phosphorus e f f e c t s upon nutrient absorption i n barley and showed that nitrogen absorption was greatest at a s o i l temper-ature of 19°C. Further studies by Power et a l . (1964) showed that optimum s o i l temperatures may decrease from 20-24 to 14-18°C as the plant develops. Tingle (1968) reported lowest barley plant s t e r i l i t y at 18°C and low main head s t e r i l i t y at 12 and 18°C, but increased s t e r i l i t y at 24 and 30°C. Optimum temperature f o r growth and development of the barley plant was found to be 18°C. I t i s evident from these studies that most barley v a r i e t i e s grow best at a i r temperatures between 16 and 20°C and that too low or too high temperatures are not s a t i s f a c t o r y f o r growth. Peas Peas are well known as "cool season" crops and of various environmental factors which a f f e c t pea y i e l d s , temperature i s of s p e c i a l importance. Beattie et a l . (1962) summarized the e f f e c t of temperature on growth and development of peas i n the following manner. Pea seeds germinate and grow vigorously at lower temperatures. Cool weather i s e s s e n t i a l for obtaining good y i e l d and a high q u a l i t y product. High temperature checks the growth of the plant and causes premature flowering. Also, high temperature increases the rate of maturity besides stunting the growth of vines and reducing the y i e l d . 19 S t a n f i e l d et a l . (1966) studied the e f f e c t of temperature on growth of peas i n c o n t r o l l e d environment cabinets and reported increased node production p r i o r to flowering as the average temperature increased. Rate of stem elongation was greatest at 21/13°C (day/night) and plant height was greatest at 16/10°C. Optimum temperature f o r dry matter accumulation was found to be between 21/10 and 16/10°C (day/night). They also found that a combination of high day and high night temperature caused an increase i n the number of nodes to the f i r s t flower. Pea y i e l d s were decreased as temperature increased above 16/10°C, mainly due to a reduction i n the number of pods per plant. Went (1957) suggested that the optimal temperatures for stem elongation decrease i n the course of development. Wang and Bryson (19 56) also observed that the optimum temperature range f o r growth of peas changed as the plant passed through various stages of development. For example, 14-24 to 18-31°C was the range f o r the f i r s t two stages and 10-21°C was the range for the l a s t two stages. Highkin (1960) observed that f l u c t u a t i n g (day/night) temperature i s necessary f o r proper growth of the pea plant. A constant day and night temperature i s i n h i b i t o r y and i s not i n agreement with endogenous diurnal rhythms which occur i n plants and animals. 20 Rape Rape i s grown as a summer crop as well as a winter . crop depending on the area and va r i e t y . Usually most v a r i e t i e s are adapted to extremes of temperature. When rape was grown at 8/16 and 1U/22°C at various daylengths, the plants showed a marked reduction i n the length of the vegetative phase with increasing daylength and high temperature (Breteschneider-Herman and Schuster, 1967). The period from f e r t i l i z a t i o n to maturity was shorter at 26.5°C than at 16 or 21°C and i t was twice as long at 10°C (Canvin, 1965). T o r s s e l l and Johnson (1963) studied the ef f e c t s of experimentally produced f r o s t on winter rape and winter turnip rape at bud and flowering stages. Cold hardiness decreased with developmental stage from bud stage to post-flowering stage when tested with temperatures ranging from -10.5 to -1.8°C. The frequency of flower abortion was greater when the plants were treated at and a f t e r the flowering stage than at e a r l i e r stages. The e f f e c t of low temperature treatment on t o t a l dry matter production was found to be less than i t s e f f e c t on seed production. Low temperatures stimulated the formation of t i l l e r s and pods but reduced the seed y i e l d per pod. Wilson (1967) measured the net as s i m i l a t i o n rate (NAR) and l e a f area r a t i o (LAR) i n three species including rape and found that most' of the v a r i a t i o n i n NAR and RGR was accounted for by v a r i a t i o n i n mean temperature and r a d i a t i o n . The increase i n NAR with temperature was lea s t i n rape. Relative growth rate increased 21 with temperature and l i g h t ; the temperature was more important p a r t i c u l a r l y i n cool weather. Rape had a temperature optimum of 24°C. The l e a f area r a t i o rose with an increase i n temperature. Low temperature, high i n s o l a t i o n and moderate r a i n f a l l are conducive to high y i e l d s of seed with a high o i l content. Canvin (1965) and D o r r e l l and Downey (1965) reported an increase i n o l e i c acid content and a decrease i n l i n o l e i c acid and erucic acid content with increase i n temperature. Mechanism of Ion Uptake The mechanism of ion uptake can be broadly categor-ized into "passive" and "active". In the former, ions may enter roots by d i f f u s i o n or through exchange adsorption and are believed to then exi s t i n the "outer space" of the root (Epstein, 1955, 1960 and Hylmo, 1955, 1958). The l a t t e r method of entry i s according to Epstein's (1955) c a r r i e r concept, i n which ions combine with c a r r i e r molecules and the r e s u l t i n g ion c a r r i e r complexes traverse membranes of l i m i t e d permeability to the free ions. At the inner surface of the membranes ions are released from the c a r r i e r . The active entry of ions, which implies the use of metabolic energy, i s supported by the work of Brouwer (1956) and Van den Honert et a l . (1955). The basic differences between "passive" and "active" absorption are outlined by Epstein and Legette (1954). 22 Lundergardh and Biirstrom (1933) were the f i r s t to suggest that s a l t accumulation i n the roots depends on aerobic r e s p i r a t i o n . They showed the necessity of oxygen access f o r s a l t uptake i n t h i n discs of storage organs, whereas Steward and Preston (1941), and Hoagland and Broyer (1942) r e s t r i c t e d themselves to proving that r e s p i r a t i o n i s a general requirement for s a l t accumulation. Subsequently Lundergardh and Burstrom (1958) reported that the relationship between r e s p i r a t i o n and s a l t uptake i s quantitative and the amount of ions taken up depends on the i n t e n s i t y of that part of r e s p i r a t i o n which i s i n h i b i t e d by cyanide. The ground respiration, almost r e s i s t a n t to cyanide,does not exercise any d i s t i n c t influence on s a l t uptake. Since r e s p i r a t i o n i s f a r more intimately r e l a t e d to the absorption of anions than to the absorption of cations i t i s known as anion r e s p i r a t i o n . Lundergardh (19 54) was able to show by spectrophotometric observations that the biochemical basis of anion r e s p i r a t i o n i s the a c t i v i t y of the cytochrome system. The oxidation-reduction status of the cytochrome system was very c l o s e l y connected with the presence or absence of s a l t s . This led to the hypothesis that there i s a s t r i c t connection between electron flow i n the r e s p i r a t o r y chain and anion movement i n the opposite d i r e c t i o n . The Lundergardh hypothesis holds that anions t r a v e l along the chain of a l t e r n a t e l y oxidized and reduced members of the cytochrome chain, being i n i t i a l l y caught by cytochrome oxidase (Bonner and Varner, 1965). 23 Although the electrochemical theory of anion r e s p i r a t i o n has much support, i t i s the c a r r i e r theory that i s i n vogue today, e s p e c i a l l y a f t e r the work of Epstein and Hagen (1952). The current concept of ion binding compounds as c a r r i e r s appears to be widely accepted (Epstein, 1953 and Thomas, 1956). In t e r i o n i c r e l a t i o n s h i p s i n s a l t accumulation have been studied and i t i s possible to demonstrate f o r example that K, Rb and Cs compete for the same s i t e whereas Na and L i do not compete f o r the same s i t e . S i m i l a r l y Epstein and Legett (1954) have found that Ca, Ba and Sr compete f o r i d e n t i c a l s i t e s and that Mg passes over d i f f e r e n t s i t e s . Also, i t has been shown that ^PO^ and HPC^ enter over d i f f e r e n t s i t e s whereas OH ions i n t e r f e r e competitively with both s i t e s . Legett and Epstein (19 56) showed that SO^ and SeO^ compete f o r the same s i t e . NOg and CI were found to have no measurable a f f i n i t y f o r the SO^-SeC^ binding s i t e . On the other hand Tanada (19 55) reported stimulatory e f f e c t s of Ca on the uptake of Rb and P without a s a t i s f a c t o r y explanation. Legett and Epstein (1956) observed a stimulatory e f f e c t of Ca on SO^ i n excised barley roots. Legett (1956) has obtained evidence which indicates that Ca acts by v i r t u e of increasing the "turn-over" rate of the s i t e involved with HP0 u absorption. 24 Although various compounds have been suggested, the chemical nature of these c a r r i e r s i s s t i l l unknown. Steward and Street (1947) postulated that phosphorylated energy r i c h N compounds function as c a r r i e r s . Tanada (19 55) favoured the ribonucleoprotein as a c a r r i e r and suggested that the nucleic acid portion binds the cations while the protein moiety binds the anions. There i s complete agreement that ion-binding compounds must be capable of undergoing some other change i n energy l e v e l s i m i l a r to that i n phosphorylated compounds (Gauch, 1957). Transport of Minerals V/ithin the Plant Ions entering the root system move predominantly i n a transverse d i r e c t i o n across the cortex toward the s t e l e and those which are not retained by intervening c e l l s are transferred into the xylem. The xylem tracheae constitute an apparently i n e r t tubing system, through which water moves with a minimum of metabolic involvement. Due to the presence of p i t s on walls and end plates of tracheids i t i s possible fo r the xylem sap to move both v e r t i c a l l y and l a t e r a l l y . The entry of inorganic materials into the xylem from the s o i l appears to be most active i n the root t i p , and appears to be metabolically regulated. Bollard (1958) reported that xylem sap consists of both minerals and organic material, and that the r a t i o of these two can change with the season. Usually the xylem sap contains organic material such as carbohydrates and nitrogenous material, and inorganic nutrients such as S, K, Mg, Ca, P and Fe. Metals are mostly i n the chelated form (Stewart, 19 63) and among anions S may move as sulphate and P as phosphoryl choline (Bollard, 1960). The upward movement of material i n the xylem has been properly established a f t e r the work of Stout and Hoagland (1939). Then Biddulph (1951) measured the amount of radioactive phosphorus accumulating i n bean leaves a f t e r 4 days and showed that the phosphate accumulates almost exponentially i n successively higher leaves. The rate of t r a n s l o c a t i o n i n the xylem i s known to be greatly influenced by the rate of t r a n s p i r a t i o n . Xylem sap nutrients move i n the t r a n s p i r a t i o n a l medium thus high rates of translocation of materials have been observed when tr a n s p i r a t i o n rates are high. Biddulph and Markle (1944) and Ray (1963) reported that some e s s e n t i a l minerals are r e d i s t r i b u t e d i n the plant tissues with remarkable f a c i l i t y . In most plants mobile elements are transferred to new leaves at the expense of old ones and t h e i r deficiency i s r e f l e c t e d on a l l parts of the shoot. The pattern of movement and degree of mobility vary with the mineral. It has been demonstrated that P moves with great ease both upward and downward from the l e a f . P i s trans-located more during the day than at night and i s moved mainly i n the metabolically active t i s s u e . S i s also mobile i n plants. In areas where i n d u s t r i a l operations discharge S into the a i r , the element enters the plant through the leaves and i s not only used i n metabolism but also i s translocated to other t i s s u e s . Biddulph et a l . (1958) observed s t r i k i n g differences i n the d i s t r i b u t i o n pattern of P, Ca and S i n bean plants. P and S were retranslocated from the primary leaves within 6 hours a f t e r exposure to tracers while elements such as Ca were not retranslocated. Sulphur i n the N u t r i t i o n of Plants  Sulphur Deficiency and the Relative Importance of Various Sources of Sulphur The existence of S as an e s s e n t i a l element f o r plant growth has been known fo r a long time, but only recently has i t received the attention that i t deserves as a plant nutrient. S d e f i c i e n c i e s have been reported from various parts of the world, and are widespread i n A u s t r a l i a , New Zealand, South America and North America, and t r o p i c a l areas of A f r i c a and Asia. In Europe they have been reported from France, Germany, Iceland, Netherlands, Norway, Finland, Sweden, Spain and Yugoslavia. As early as 1900 S d e f i c i e n c i e s were observed i n the P a c i f i c Northwestern United States and i n Central states such as Nebraska and Minnesota (Anon., 1967). S deficiency was f i r s t discovered i n Canada i n 1927 on grey wooded s o i l s i n the province of Alberta (Wyatt, 1945). In 193 8 d e f i c i e n c i e s were reported i n Saskatchewan and i n 1947 they were observed on the grey wooded s o i l s of the c e n t r a l i n t e r i o r of B r i t i s h Columbia (Beaton et a l . , 1966). S d e f i c i e n c i e s occur as a r e s u l t of the increased use of e s s e n t i a l l y S-free f e r t i l i z e r s , increased crop y i e l d s thereby 27 requiring a l l of the e s s e n t i a l plant nutrients i n larger amounts; decreased use of S as fungicides and i n s e c t i c i d e s ; and increasing s u b s t i t u t i o n of natural gas by other refine d f u e l o i l s (Coleman, 1966). At the time when such f e r t i l i z e r s as superphosphate and ammonium sulphate were used, S d e f i c i e n c i e s were not observed due to the large percentage of S added i n c i d e n t a l l y to the s o i l along with other elements. However, at present high analysis f e r t i l i z e r s are added to the s o i l as they are a t t r a c t i v e to the manufacturer and to the grower because of economics of production, transportation and handling and ap p l i c a t i o n costs. As a r e s u l t sulphur removed by crops and by leaching i s not being replaced i n the s o i l (Burns, 1967 and Howard and Ensminger, 19 5 8). For many years S was used f o r the control of various plants and animal pests. However, at the present time very small amounts of S are used as a p e s t i c i d e , due to the a v a i l -a b i l i t y of synthetic o r g a n i c materials that are more e f f e c t i v e against pests, and the contribution from t h i s source i s small. Of the various sources of S to the s o i l , the most important are the o r i g i n a l supply from parent material, plants and animals, and the atmosphere. The lithosphere contains about 0.06 per cent S (Howard and Ensminger, 1958; Tisdale and Nelson, 1966). It i s o r i g i n a l l y found as the sulphide of metals i n plutonic rocks. Tisdale and Nelson (1966) reported that when these rocks decompose due to weathering processes the 28 sulphide i s oxidized and i s released as sulphate. The sulphates are then p r e c i p i t a t e d as soluble and insoluble sulphate s a l t s i n a r i d or semi-arid climates. Sulphur dioxide of the a i r may pass d i r e c t l y into the l e a f t i s s u e of the growing plant or part of i t can be brought down with the r a i n and accumulate i n the s o i l . Olsen (1956) worked on cotton and reported that sulphur dioxide was absorbed by the growing plants d i r e c t l y from the atmosphere and about 3 0 per cent of the plant requirement was obtained i n t h i s manner. The main sources of atmospheric S according to Swanson and Whitney (1968) are from sea spray, H 2S and v o l a t i l e sulphide released from marshlands and the sea, and sulphur dioxide released from the burning of sulphurous f u e l s . Larger amounts from these sources are availa b l e from metropolitan areas than from r u r a l areas f o r plants, as reported by Seim et a l . (1968). I t i s d i f f i c u l t to assess the av a i l a b l e S i n the s o i l as i t i s complicated by the m u l t i p l i c i t y of sources of availa b l e forms and t h e i r magnitude i n r e l a t i o n to crop requirements. Tisdale and Nelson (1966), Reisenauer (1967) and Williams and Steinberg (1964) reported that S i s found i n arable land i n the form of soluble sulphate i n the s o i l s o l u t i o n , i n the form of organic matter, or adsorbed on the s o i l complex. Freney et al_. (1962) reported that the major portion of S i s avai l a b l e i n the upper layers i n organic combination and that sulphates dominate at lower layers. L i t t l e i s known of the 29 constituent compounds of the organic form. Lowe and de Long (1961) showed evidence that sulphate S was associated with the organic f r a c t i o n of the s o i l . Lowe (1964) studied the S status of f i v e Quebec s o i l s and reported that S i n these s o i l s was mainly organic i n nature and that organic sulphates were dominant i n mineral s o i l s , whereas organic sulphate and carbon bonded S fr a c t i o n s were of s i m i l a r magnitude i n the organic s o i l s . They also found a greater amount of adsorbed sulphate i n mineral s o i l s than i n organic s o i l s . Also Lowe (1965) studied the nature and d i s t r i b u t i o n of S fr a c t i o n s of selected Alberta s o i l p r o f i l e s and found that the t o t a l S content of surface s o i l increased from the brown s o i l zone to dark brown to black and f e l l o f f again i n the dark grey and grey wooded zones. Soluble sulphates have been shown to be variable and to reach high l e v e l s i n the lower horizons. The C:N:S r a t i o of the organic f r a c t i o n has been measured i n a wide var i e t y of s o i l s and has been found to be approximately 125:10:1.2 f o r various crops (Reisenauer, 1967). Tisdale and Nelson (1966) reported 113:10:1.3 for calcareous s o i l s and 147:10:1.4 for the humid non-calcareous s o i l s . The r a t i o s of N:S i n a l l studies were found to be quite uniform. Sulphate Sulphur i n S o i l s Although SO^ may account f o r less than 10 per cent of the t o t a l S i n surface s o i l s , the amounts i n subsoils may represent a s i g n i f i c a n t portion of the t o t a l . The SCv i n the 30 s o i l s olution i s i n equilibrium with s o l i d phase forms, both that adsorbed on the sesquioxide of acid s o i l s and that present as s l i g h t l y soluble forms. The amount, rate of renewal, d i s t r i b u t i o n and losses of these forms from the rooting zone dominate the S nutrient status of the s o i l (Reisenauer, 1967). The movement of S0l+ i n the s o i l depends on i t s concentration i n the s o i l s o l u t i o n , i t s reaction with the s o l i d phase components, and v e l o c i t y and amount of water. Swoboda and Thomas (1965) reported that a r a i n f a l l of over HO inches could move SO^ to lower layers. In a r i d s o i l s SO^ behaviour i s dominated by the s o l u b i l i t y of gypsum (Dutt and Anderson, 1964). According to Bardsley and Kilmer (1963), the amount of SO^ retained by s o i l s depends on pH. The amount retained by acid soils, increases with the content of hydrous oxides and clay. Kamprath et a l . (1956) and Ensminger (1954) also reported that SO^ adsorption i s strongly dependent upon pH and the equilibrium SO^ concentration, with retention increasing as the pH decreases and the concentration of SO^ i n s o l u t i o n increases. Adsorbed SO^ i s i n k i n e t i c equilibrium with the SO^ i n solution and may be replaced by other anions of greater co-ordinating a b i l i t y according to the s e r i e s ; hydroxyl > phosphate > sulphate = acetate > n i t r a t e = chloride (Bingham et a l . , 1965 and Chao, 1964). Harward and Reisenauer (1966) reported that SO^ adsorption has two important roles i n plant n u t r i t i o n . F i r s t l y , the adsorbed S serves as a source of a v a i l a b l e sulphur to plants 31 and secondly i t helps to retard the SO^ movement within the s o i l , thus reducing leaching losses and modifying the d i s t r i b u t i o n of the element within the p r o f i l e . Several workers including Chao e_t al_. (1962) have reported that removal of Fe, A l and organic matter s i g n i f i c a n t l y reduced the amount of SO^ retained by s o i l s . Subsequently i n 19 6 5 Chao et_ al_. confirmed that SO^ i s retained by an anion exchange mechanism and that such a reaction i s r e f l e c t e d by an increase i n OH concentration i n the solution phase. Sulphur Oxidation i n S o i l s Oxidation-reduction reactions involving S are important i n s o i l systems. For many years there were two schools of thought as to the process by which S i s oxidized i n the s o i l . One emphasized inorganic chemical oxidation and the other b i o l o g i c a l oxidation. Sulphides, elemental S and thiosulphate can be oxidized slowly i n the s o i l by non-biological means and i n f a c t under favourable conditions elemental oxidation can proceed quite r a p i d l y . Oxidation of p y r i t e i s quite slow and needs high a c i d i t y to break down the structure and dissolve the p y r i t e (Burns, 1967). Both sulphurous acid and i t s sulphite s a l t s are active reducing agents and i n the process are themselves oxidized. S O 2 could be oxidized i n a i r to SO^ but the rate of oxidation i s known to be extremely slow at low temperatures. Tisdale and Bertramson (1949) studied the r e l a t i o n -ships between Mn and S and suggested that although a low pH 32 i s required, the c o n t r o l l i n g factor i s a supply of electrons and the oxidation of S could supply electrons f o r reduction of Mn. According to Moorman (1963) oxidation-reduction reactions involving S compounds occur i n the formation of "Cat clays". Marine sediments are neutral to s l i g h t l y a l k a l i n e when submerged but are highly acid a f t e r drainage, and t h i s has been a t t r i b u t e d to the reduction of SO^ i n sea water and the oxidation of the sulphides upon reclamation of the sediments. Other possible examples of non-biological oxidation of S could be c i t e d , but favourable conditions for many of these reactions seldom i f ever e x i s t i n s o i l s . On the, other hand favourable conditions f o r b i o l o g i c a l oxidation of S and S compounds frequently e x i s t . Inorganic chemical changes are i n s i g n i f i c a n t i n comparison with the microbial conversions. B i o l o g i c a l oxidation of S proceeds continuously i n arable s o i l s . Only such adverse conditions as severe drought or freezing could slow down or stop the a c t i v i t y of the many autotrophic and heterotrophic microorganisms. Jordan (1957) reports that i f a s o i l i s well supplied with organic matter, S oxidation i s most l i k e l y to occur. Starkey (1950) reported that when reduced forms of S are added to the s o i l i n large quantities e i t h e r i n f e r t i l i z e r s or from the atmosphere, a rapid increase i n number of S o x i d i z i n g organisms often occurs. This observation has been further supported by Moser and Olsen (1953). The autotrophic bacteria of the genus T h i o b a c i l l u s have been studied i n more d e t a i l than other S o x i d i z i n g 33 organisms and are generally considered to be the most important i n s o i l s . These organisms derive the energy for the reduction of CO^ from the oxidation of S. They can l i v e i n an environment e n t i r e l y free from organic matter and other organisms (Lees, 1955; Fry and Peel, 1954). Heterotrophic b a c t e r i a , fungi and actinomycetes are unable to form protein and carbohydrate from inorganic C and N. They are more numerous than the t h i o b a c i l l i and are more d i v e r s i f i e d than autotrophs. They require organic material as a source of energy and carbon. Due to the dependency on other organisms, t h e i r response i n terms of S oxidation can be modified tremendously (Burns, 1967). Several environmental factors have an e f f e c t on S oxidation. Optimum temperatures f o r oxidation are not the same for a l l organisms, but temperatures between 27 and 40°C are known to include most types according to Parr and P r i s t (1953) and Rudolfs (1922). Other factors such as s o i l pH and amount of CaCO^ are important when dealing with oxidation. Tisdale and Nelson (1966) reported that T h i o b a c i l l u s thioxidans i s capable of surviving at extremely low pH values. Other S ox i d i z i n g organisms had d i f f e r e n t pH requirements, but i n general oxidation of added S proceeded more r a p i d l y i n more acid s o i l s . 34 Sulphur Deficiencies and Diagnostic Techniques for  Determining S Deficiencies i n Crops and S o i l s S d e f i c i e n c i e s have been reported f o r various crops by numerous authors and the importance of S as an e s s e n t i a l element has been known from the time of L i e b i g (Coleman, 1967). Symptoms of S deficiency are quite well defined. C h a r a c t e r i s t i c a l l y they are s i m i l a r to those caused by nitrogen deficiency. Leaves i n some species l i k e rape show i n t e r v e i n a l c h lorosis (Maraby, 1968). Howard and Ensminger (19 58) reported that c h l o r o s i s i n the cotton plant may involve the whole plant or i t may be severe only on the younger leaves. Anderson and F u t r a l (1966), also working on cotton, reported symptoms of yellow c h l o r o s i s and stunted growth. U l r i c h and Hylton (1968), working on I t a l i a n ryegrass, suggested that S i s not a part of c h l o r o p h y l l , but•chlorosis has been att r i b u t e d to impairment of photosynthesis by an i n d i r e c t e f f e c t on the protein l e v e l and the c h l o r o p h y l l content of the chloroplast. Ergle (1953) found protein S of the chloroplast to be as highly correlated with the o p t i c a l density of chlorophyll extracts as protein-N. The protein-N content of the chloroplast was ten times as great, however, as that of protein-S. On most plants, young leaves are c h l o r o t i c and on others such as tobacco, c i t r u s and cotton some of the older leaves may be affected, f i r s t . Other symptoms are that plants grow small and spindly with short slender s t a l k s . Growth 35 rate i s retarded and maturity often delayed, p a r t i c u l a r l y with cereal grains. On legumes nodulation i s frequently reduced. F r u i t maturity i s delayed or does not f u l l y occur and usually a high percentage of aborted seeds are present. Anthocyanin pigmentation develops i n some plants with severe S deficiency. According to H i l d e r and Spencer (1954) Medicago species develop purple and brown t i n t s . I f a small amount of S i s supplied, these disappear and the color changes to t y p i c a l c h l o r o s i s . U l r i c h and Hylton (1968), working on I t a l i a n ryegrass, found that upper leaves of severely d e f i c i e n t plants showed a reddish-purple colour that developed mostly on the under surface of the blade and along the margin. Anderson and Spencer (1950) reported that i n white clover, roots comprised a lower proportion of t o t a l plant weight i n S s u f f i c i e n t plants. With no S a high percentage of roots was obtained, mainly due to poor top growth. Diagnostic techniques f o r determining S d e f i c i e n c i e s i n crops and s o i l s have been studied by several workers. Ensminger and Freney (19 65) have outlined the diagnosis of S deficiency by plant and s o i l a n a l y s i s . In plant analysis these workers have suggested a scheme i n which the concentration of S i n the plant should be at a " c r i t i c a l percentage" above which there i s luxury consumption and below which there i s poverty adjustment. The " c r i t i c a l percentage" i s normally determined by t r i a l and error. The use of t h i s method was f i r s t discussed by U l r i c h (1952). 36 The " c r i t i c a l percentage" should be the same f o r each species over a wide var i e t y of s o i l types and c l i m a t i c conditions. Harward et a l . (1962) reported a " c r i t i c a l percentage" of 0.20 per cent S f o r a l f a l f a , and U l r i c h and Hylton (1968) reported 100 ppm SO^-S i n l e a f t i s s u e of ryegrass. Total S, SO^-S and the S:N r a t i o have been used for detection of S deficiency i n plants. Cairns and Carson (1961) and Cressman and Davis (1962) reported a d i r e c t r e l a t i o n s h i p between t o t a l S l e v e l s i n plants and S supply. Although several workers have used t o t a l S l e v e l s i n plant tiss u e as an i n d i c a t i o n of S deficiency or s u f f i c i e n c y , i t i s generally recommended that SO^-S i s a better measure, not only because of the ease with' which an analysis f o r SO^-S can be performed but also because i t works better than t o t a l S for many species. U l r i c h and Hylton (1968) observed the same l e v e l of t o t a l S i n the stem of ryegrass when S Was supplied at d i f f e r e n t l e v e l s , but the SO^-S l e v e l s showed a nutrient y i e l d r e l a t i o n highly desirable f o r diagnostic purposes. Ensminger and Freney (1965) suggested that absorbed SO^-S i n plants i s reduced to other forms during metabolism, and where the S i s i n short supply most of the S i s incorporated into protein and l i t t l e SO^ can be detected i n the plants. SO^-S l e v e l s have been shown to be r e l a t e d to the S status of s o i l s f o r sugar beets as reported by Walker 37 and Bentley (1961), for I t a l i a n ryegrass by U l r i c h and Hylton (1968), and for several other species by many other workers. However, Anderson and Spencer (1950b) have cautioned that SO^ could accumulate i n the plant i f any deficiency a f f e c t i n g protein synthesis, such as Mo, i s present. N deficiency could also cause a higher concentration of SO^. It i s necessary to standardize the part of the plant to be analyzed. U l r i c h e_t a l . (1959) found that the sugar beet l e a f blades are better indicators than the p e t i o l e s , as there was a wider range of values f o r SO^-S between deficiency and s u f f i c i e n c y . Biswas and Sen (1959) reported high a c t i v i t y i n the lower leaves of 10 day old pea plants compared with 35 upper leaves when fed with SO^ l a b e l l e d with S Age of the plant has d e f i n i t e e f f e c t s on the concentration of SO^ and t o t a l S i n some'plants, and should be considered when diagnosing S d e f i c i e n c i e s as reported by Ensminger and Freney (1965). Other factors such as temperature could a f f e c t the S concentration i n plants tissue (McKell and Wilson, 1963). Also, residues from f u n g i c i d a l and i n s e c t i c i d a l sprays could a f f e c t the plant analysis r e s u l t s . It has not been possible to make any extensive use of s o i l - S values as diagnostic c r i t e r i a . The l i m i t a t i o n i n information r e s u l t s not from lack of past i n t e r e s t but rather from the fact that extraction procedures are currently undergoing c r i t i c a l analysis and development, with the r e s u l t 38 that standardized data are currently l i m i t e d (Eaton, 1966). The uncertainties which have existed i n the i n t e r p r e t a t i o n of' s o i l analysis as regards a v a i l a b i l i t y point to the advantage of tissue analysis f o r purposes of diagnosis. Sulphur Requirements f o r Various Crop Species Generally crops require about the same amount of S as they do of P. Crop plants of the Cruciferae and L i l i a c e a e f a m i l i e s have p a r t i c u l a r l y high S requirements and legume crops require more than small grain, grasses and corn (Beaton, 1966 and Eaton, 1966). Crop requirements f o r the response to S f e r t i l i z a t i o n are very much dependent upon the l e v e l of production. Beaton (1966) has made an extensive survey of the crops that have responded to S f e r t i l i z a t i o n i n Canada, U.S.A. and several other parts of the world. Beaton et_ a l . (1966) also studied the response of several legumes and legume-grass mixtures to S f e r t i l i z a t i o n i n various parts of B r i t i s h Columbia and observed responses on grey wooded s o i l s i n the c e n t r a l i n t e r i o r , grey wooded and grey forested s o i l s i n the north Okanagan and brown wooded s o i l s i n the east Kootenay. In Alberta too, most of the y i e l d increases due to S a p p l i c a t i o n were obtained on grey wooded s o i l s , but increases were also observed on a few podzolic and chernozemic s o i l s . S i m i l a r l y i n the provinces of Saskatchewan and Manitoba responses to S applications were observed on grey wooded s o i l s . Maraby (1968) worked on the S deficiency of the rape crop i n France and recommended a f e r t i l i z e r program for winter rape 39 based on four elements, namely N, P, K and S. U l r i c h and Hylton (1968) measured the growth and mineral content of I t a l i a n ryegrass and reported the requirement of high f e r t i l i z e r f or persistence and to use lim i t e d r a i n f a l l e f f i c i e n t l y . They also observed a r e l a t i o n s h i p of plant growth to S concentration and to S supply i n plant parts. Seim et a l . (1969) reported increased content of S i n a l f a l f a t i s s u e when the rate of a p p l i c a t i o n to the s o i l was increased. Their values corroborated the findings of other workers. They found values ranging from .07 to .5 per cent S and they assigned .3 per cent as the l e v e l i n d i c a t i v e of adequate supply. Cairns and Carson (1961) reported S concentration of .14 to .17 per cent i n untreated a l f a l f a . Jordan and Bardsley (1958) reported the c r i t i c a l S l e v e l i n f i e l d grown clover to be between .10 and .16 per cent. Nitrogen-Sulphur Ratios i n Evaluating Sulphur Status of  Various Species Several workers have employed the N:S r a t i o to evaluate the S status of various crop species. Walker e_t a l . (1956) worked on the N:S r a t i o of clover. Pumphrey and Moore (1965) showed the value of the N:S r a t i o i n p r e d i c t i n g the S status of 22 a l f a l f a f i e l d s . Values below a N:S r a t i o of 11 indicated adequate supply, while higher values of 15 to 25 indicated progressively more acute d e f i c i e n c i e s of S. Stewart and Porter (1969) studied the N:S r a t i o s i n the tops and roots of wheat, corn and beans i n r e l a t i o n to N and S supply i n the s o i l . I t was shown that when S became l i m i t i n g , a d d i t i o n a l N did not a f f e c t e i t h e r the y i e l d or protein l e v e l of the plants, but the non-protein N increased. Their data also indicated that one part of S i s required f o r every 12 to 15 parts N to ensure maximum production of both dry matter and protein. Sorensen et_ a l . (1968) studied the S content and y i e l d of a l f a l f a i n r e l a t i o n to plant N and S f e r t i l i z a t i o n , and reported a close r e l a t i o n s h i p between S and N i n a l f a l f a grown with and without applied S on two d i f f e r e n t types of s o i l s . Stewart (1966) worked on wheat and sugar beet and found that N:S r a t i o s are useful i n assessing S d e f i c i e n c i e s . .The N:S r a t i o s of the protein of both species were s i m i l a r , averaging 15.1 f o r wheat and 16.6 f o r sugar beets. When no S f e r t i l i z e r was added to S d e f i c i e n t s o i l s , the forage produced had N:S r a t i o s greater than 17. Nitrogen and Sulphur Interactions i n Plants It has been known that S deficiency causes profound changes i n the N metabolism of plants with reduced protein synthesis and accumulation of soluble organic and inorganic nitrogenous compounds. This change i n nature of nitrogenous compounds i s probably r e l a t e d to the i n a b i l i t y of the plant to reduce n i t r a t e which i s e s s e n t i a l f o r the formation of organic nitrogen compounds and proteins. Eckerson (1932) has reported that sulphur d e f i c i e n t tomato plants had less n i t r a t e reductase than normal plants, which could explain the 41 increased NOg-N content. Walker (1957) also reported NO^-N accumulation i n S d e f i c i e n t non-legumes, because of i n s u f f i c i e n t protein synthesis and subsequent lack of growth. U l r i c h and Hylton (1968) observed an increase i n phosphate-P and t o t a l P i n the f i r s t blade of S d e f i c i e n t ryegrass. The phosphate-P f r a c t i o n constituted about 7 7 per cent of the t o t a l P concentration of the f i r s t blade of non-d e f i c i e n t plants. This proportion was raised to 100 per cent of the t o t a l P f o r S d e f i c i e n t plants. I t was postulated that S deficiency interrupted phosphate-P metabolism to a greater extent than that of nitrate-N. According to Allaway and Thompson (1966) the S amino acid' content of normal plants was higher than that of the "minus S" plants due to the fact that the normal plants synthesized more protein. Generally, S adequate plants contain more S amino acids than S d e f i c i e n t plants. It i s reported that the increase i n concentration of S amino acids takes place through an " a l l or none" process c o n t r o l l e d by the synthesis of plant protein. The amount of d i f f e r e n t amino acids per unit of protein tends to be f a i r l y constant. Therefore species or v a r i e t i e s that are capable of synthesizing more proteins high i n S amino acids could be grown successfully under optimum conditions. Several workers have studied the e f f e c t of S f e r t i l i z a t i o n on protein q u a l i t y . Saalbach (1962) described pot experiments with barley which showed that 42 methionine content i n barley grains f e l l by 9 per cent and the cysteine content by 3 8 per cent under conditions of S deficiency. Sulphur Metabolism General Aspects S i s one of the three important anions involved i n plant n u t r i t i o n along with N and P. I t i s important as a s t r u c t u r a l component as well as a p a r t i c i p a n t i n the resp i r a t o r y metabolism. Chemically S behaves l i k e N and exists i n valency states ranging from highly p o s i t i v e (+6) to negative (-2). S also occurs i n widely d i f f e r e n t combinations, as with other S atoms (polysulphides), with oxygen atoms (sulphates, e t c . ) , and with both S and oxygen atoms (thiosulphates, e t c . ) . Due t'o i t s existence i n diverse valency states and the m u l t i p l i c i t y of compounds'formed, i t i s d i f f i c u l t to study the metabolism of S i n plants and .microorganisms and as a r e s u l t knowledge has lagged behind C, N and P metabolism (Thompson, 1967). The reduction of SO^ to the SH (sulphhydryl) l e v e l and i t s incorporation into the amino acids, peptides and proteins i s centered around the formation of the amino acids cysteine and methionine. A l l animals and some microorganisms are ultimately dependent upon plant and microbial sources of cysteine and methionine (Gibbs and S c h i f f , 1960). Considerable progress has been made during the l a s t decade 43 with regard to the enzymatic studies of several unrelated microorganisms. These studies have revealed some of the reactions and intermediates involved i n the SO^ metabolism of the organisms. Similar studies also have been i n i t i a t e d with the higher plants and basic s i m i l a r i t i e s have been observed. Role of Sulphur Compounds i n Structure and Their Function  i n Metabolism S compounds play an important r o l e i n protein, and both cysteine and methionine are associated with protein structure. Proteins function i n nature as s t r u c t u r a l e n t i t i e s , as c a t a l y s t s , as oxygen c a r r i e r s , as hormones, and i n several other ways. Allaway and Thompson (1966) reported that S amino acids provide disulphide (-S-S-) bonds from cysteine for the covalent cross linkage of two peptide chains or f o r the formation of stable loops i n a single chain. The sulphydryl (-SH) groups provide s i t e s for attachment of m e t a l l i c cations, sulphydryl bonds may also function as s i t e s of hydrogen bonding, and as points of attachment of prosthetic groups to enzymes. White (1960) showed the importance of disulphide bonds i n the e f f e c t of reduction upon the enzyme ribonuclease. Destruction of disulphide bonds by c a r e f u l reduction removes enzymic a c t i v i t y , and by oxidation enzymic a c t i v i t y can be restored to reform the disulphide bonds. 44 Metabolically the function of S i s important and varied. Sulphydryl groups on enzyme proteins serve as the s i t e of binding of the substrate to the enzyme. This i s made possible through a thioester bond. Wilson et a l . (1961) showed evidence for a protein that transfers hydrogen by oxid-ation of sulphydryl groups and reduction of disulphide groups. S i s important i n the biosynthesis of f a t t y acids from a c e t y l groups and i s made possible by binding a l l the intermediates to one protein (Vagelos, 1964 and Conn and Stumpf, 1966). S compounds that function as a c e t y l c a r r i e r s i n the oxidation of a keto acids are thiamine, l i p o i c acid and coenzyme A. In the oxidation of pyruvate, a c e t y l coenzyme A i s one of the products. Acetyl coenzyme A then forms the substrate f o r the formation of a number of other compounds, such as c i t r i c a c i d , acetoacetic acid, malonic a c i d , etc. (Allaway and Thompson, 1966; Conn and Stumpf, 1966 and Bonner and Varner, 1965). According to Allaway and Thompson (19 66) thiamine does not bind acyl groups d i r e c t l y to S, but i t seems quite l i k e l y that the S i n the molecule i s responsible f o r the unique r e a c t i v i t y of the carbon that serves as the s i t e of attachment of the acyl group. Thiamine also functions i n the transfer of two carbon f r a c t i o n s of keto sugars to sugar phosphates (transketolation) (Bonner and Varner, 1965). 45 B i o t i n i s involved i n the metabolism of the products of keto acid oxidation and acts as a carbon dioxide c a r r i e r i n carboxylation reactions. According to Mistry and Dakshinamurti (1964), functional b i o t i n i s bound to l y s i n e i n protein through a peptide bond. Methionine i s known to play an important r o l e i n one carbon transfer reactions. It i s the p r i n c i p a l n a t u r a l l y occurring transmethylating agent (Meister, 1965). Allaway and Thompson (1966) reported that glutathione, a t r i p e p t i d e , i s ubiquitous i n l i v i n g organisms and i t s most probable function i s the maintenance of appropriate oxidation-reduction conditions i n c e l l s . Carbon Dioxide Exchange Photosynthesis Photosynthesis rates depend on the i n t e r p l a y of a l l the interdependent environmental and i n t e r n a l factors that mutually a f f e c t the o v e r a l l process. Light and the concentration of CC^ are the two external factors most d i r e c t l y concerned, but photosynthesis i s also influenced by a l l the other environmental factors that a f f e c t the proper functioning of the plant (Thomas, 1965). A few of these factors pertinent to the present study w i l l be discussed. Rabinowitch (1956) stated that under a l l conditions, the rate of photosynthesis changes with temperature, excepting under l i g h t - l i m i t e d conditions i n which the v e l o c i t y of the o v e r a l l process i s equal to that of the primary photochemical 46 reaction. He also reported that the optimum temperature for land plants i s around 30-35°C and that a decline i n photo-synthetic rates at higher temperatures i s p a r t l y caused by a rapid r i s e i n r e s p i r a t i o n . At high temperature thermal inhib-i t i o n i s caused by a destructive process and i s not associated with an i n t r i n s i c property of the k i n e t i c mechanism of photosynthesis. At low temperatures v i s c o s i t y increases and slows down the d i f f u s i o n of CC>2 thereby causing a CC^ li m i t e d state. Lundegardh (1966) reported that the c o n t r o l l i n g e f f e c t of temperature on photosynthesis applies more to the chemical than to the purely physical processes; the l a t t e r depend on the biochemical reactions. If such processes as CC^ consumption, production and sugar synthesis are slower, then an increase i n temperature would have a greater e f f e c t on a l l the processes. He therefore suggests that together with the chemical p a r t i a l processes, physical processes such as solution of CC^ and 0^ , and t h e i r speed of d i f f u s i o n also come into play. Wilson (1966) found that maximum net a s s i m i l a t i o n rates f o r rape and maize, varied with temperature between 12 and 30°C for rape and 23 and 36°C f o r maize. Baldry e_t a l . (1966) determined the e f f e c t of temperature on photosynthesis i n i s o l a t e d chloroplasts under saturated l i g h t and CC^ conditions. Increase i n temperatures between 5 and 3 0°C increased the maximum rate; above 20°C values of Q. n were less 47 than 2 and below 2 0 C they were greater than 2, and increased progressively.with decreasing temperatures. Certain algae are known to grow at high temperatures. Inman (1940) and Kemper (1963) have shown evidence to support e a r l i e r reports that Yellowstone National Park thermal algae can photosynthesize at 65 to 75°C. Lundergardh (1966) reported a s l i d i n g temperature optimum f o r leaves of potatoes, cereals and several other plants with a normal CC^ concentration and strong l i g h t . He found an optimum temperature of 2 0°C, with weak l i g h t t h i s temperature optimum was s h i f t e d to 10°C and with strong l i g h t and high CC^ content i t was s h i f t e d to about 30°C. Ormrod (1961) studied the i n t e r a c t i o n of l i g h t and temperature i n r i c e and reported that a net gain of CC^ uptake could be expected at temperatures as low as 4 0°F even under low l i g h t i n t e n s i t y . According to him, periods of low l i g h t i n t e n s i t i e s are not as serious i f the temperatures are maintained low, but extended periods of high temperatures with low l i g h t i n t e n s i t i e s would be more detrimental. Therefore i t i s a general ru l e that the optimum temperature of apparent photosynthesis i s s h i f t e d upward as l i g h t and CC^ factors increase and the optimum temperature moves downward when these factors decrease. Respiration P h y s i o l o g i c a l l y active c e l l s of most plants cease r e s p i r a t i o n just below 0°C, however, i f the plants are hardened to low temperatures or i f the seeds are dried to prevent 48 freezing damage r e s p i r a t i o n can take place at lower temperatures. Respiration has been detected i n cold hardened winter cereals even at -7°C. The upper l i m i t f o r r e s p i r a t i o n i s determined by the thermostability of the cytoplasmic proteins and with the exception of thermophilic species, most plants die between 4 5 and 55°C (Langridge and McWilliam, 1967). Respiration becomes increasingly dominant at higher temperatures, and at low temperatures r e s p i r a t i o n drops more quickly than photosynthesis (Rabinowitch, 1956). Decker (1955) measured r e s p i r a t i o n at high temperatures and found that the drop i n net a s s i m i l a t i o n was too great to be explained by increasing r e s p i r a t i o n alone, as the photosynthetic process becomes heat inactivated at temperatures at which r e s p i r a t i o n i s s t i l l occurring at a rapid rate. It had been reported by e a r l i e r workers that r e s p i r a t i o n i n the l i g h t i s the same as that i n the dark. Decker (1955) reported that a f t e r a period of i l l u m i n a t i o n of tobacco leaves, the i n i t i a l rates of CC^ evolution i n darkness were about f i v e times higher than that of those observed a f t e r 6-8 minutes. Therefore he suggested that r e s p i r a t i o n was f a s t e r when there i s i l l u m i n a t i o n than when i t i s dark. Moss (1966) also reported s i m i l a r r e s u l t s for several species. Tregunna et a l . (1961) also found that when tobacco leaves are placed i n darkness a f t e r a period of i l l u m i n a t i o n the CC^ production did n o t . s e t t l e to a steady state at once, but instead had two bursts. The f i r s t was 49 short though the rate of CO^ evolution at i t s peak was high; the second lasted longer though the rate at i t s peak was much lower. The same group i n 1964 found further e f f e c t s of l i g h t on r e s p i r a t i o n during photosynthesis by working with chloro-p h y l l - r i c h and c h l o r o p h y l l - d e f i c i e n t leaves of soybeans, peperomia and corn. Forrester et a l . (1966) studied the e f f e c t of O2 on photosynthesis, photorespiration and r e s p i r a t i o n . They observed that the rate of apparent photosynthesis was in h i b i t e d by 0 2 while the rate of r e s p i r a t i o n a f t e r a few minutes i n the dark was not affected and suggested that part of the i n h i b i t i o n of apparent photosynthesis was a r e s u l t of increased photorespiration. This stimulation of photo-r e s p i r a t i o n was manifested by an increase i n CC^ compensation point. Tregunna et a l . (1966) reported that the d i f f e r e n t i a l e f f e c t of C>2 on photorespiration and dark r e s p i r a t i o n was an in d i c a t i o n that these two are d i f f e r e n t processes. Forrester et a l . (1965) observed that corn leaves did not produce CC^ i n the l i g h t at any 0^ concentration as shown by zero compensation point and by the absence of a CC^ burst i n the f i r s t minute of darkness. Moss (1966) and others have reported quantitative differences i n photosynthesis i n d i f f e r e n t species. Maize, sugar cane and sorghum have rates of photosynthesis i n high l i g h t two to three times as great,as sugar beet, orchard grass and several other species. Thus, some e f f i c i e n t species have very low C0 9 compensation concentrations, usually 5 ppm or l e s s , 50 and do not exhibit l i g h t r e s p i r a t i o n as reported for other plants. Downton and Tregunna (1967) suggested that plants with Calvin's carboxylation reactions had high values and that plant with Hatch and Slack (Hatch and Slack,1966; Hatch et a l . , 1966 and Slack and Hatch, 19 67) photosynthetic carboxylation pathways had low compensation points. Further, the l a t t e r group of plants had well developed parenchyma bundle sheaths containing a high concentration of chloroplasts which accumulat large amounts of starch. They also reported a temperature optimum fo r photosynthesis of about 3 5°C f o r low compensation plants and 10-25°C f o r high compensation plants. Sulphur and Photosynthesis Most of the factors involved i n plant vigour are l i k e l y to be r e f l e c t e d i n the rate of photosynthesis, yet net a s s i m i l a t i o n rate per unit area of l e a f surface tends to be constant (Thomas, 1965), even by plants that d i f f e r widely i n si z e and vigour. High y i e l d s are obtained by supplying water and nutrients, mainly by changing the a v a i l a b l e l e a f area f o r photosynthesis. Many n u t r i t i o n a l d e f i c i e n c i e s could reduce photosynthesis as many elements are involved i n phases of photosynthesis. Thomas et a l . (1943) grew a l f a l f a i n sand culture pots using nutrient solutions with d i f f e r e n t l e v e l s of sulphate and observed c h l o r o t i c symptoms of leaves at the lowest l e v e l s (0.3-0.6 ppm S). These plants had reduced photosynthetic rates 51 as compared with plants supplied with 1.6 ppm S or more. As discussed e a r l i e r , S p a r t i c i p a t e s i n many of the enzymatic reactions i n the le a f through l a b i l e sulphydryl, and S groups. It i s also a constituent of the amino acids cysteine, cystine and methionine, the peptide glutathione and the vitamins, thiamine, b i o t i n and others. Calvin (1956) suggested that the transfer of electrons from l i g h t excited chlorophyll to TPNH i s accomplished by an intermediate reduction of the disulphide form. He also stated that the acetyl a t i n g coenzyme A which mediates a pyruvic acid oxidase reaction between the photo-synthetic cycle and Krebs cycle contains sulphydryl, disulphide and possibly t h i o z o l i n e groups. However, Arnon et a l . (1958) did not support t h i s theory. Hansen et al_. (1941) reported that chloroplast proteins are r i c h i n S and contain approximately 7 0 per cent of the t o t a l protein S. They also found that the r a t i o of chloroplast protein N/chloroplast protein S remained approx-imately constant during the l i f e cycle of sudan grass. Ergle (1953) reported reduced l e v e l s of c h l o r o p l a s t i c protein N and protein S and chlorophyll due to N and S defi c i e n c y i n cotton plants. Although S i s not a part of c h l o r o p h y l l , c h l o r o s i s has been a t t r i b u t e d to the impairment of photosynthesis by an i n d i r e c t e f f e c t on the protein l e v e l and chlo r o p h y l l content of the chloroplast. 5 2 Growth Stages and Photosynthesis The growth stage at which photosynthesis i s measured i s important f o r many species; the rate of photosynthesis changes with the growth stage. Tanaka and Yamaguchi (196 8) reported that at early growth stages, net as s i m i l a t i o n rate i s high and the growth e f f i c i e n c y i s about 6 0 per cent because r e s p i r a t i o n rate i s f a i r l y high and as s i m i l a t i o n products are e f f i c i e n t l y u t i l i z e d f o r growth. During these growth stages, r e s p i r a t i o n becomes a rate l i m i t i n g f a c t o r . A high temperature and adequate supply of nutrients accelerates r e s p i r a t i o n and re s u l t s i n a high growth rate . At l a t e r growth stages high nitrogen increases the size of organs such as leaves and causes mutual shading, thereby r e s p i r a t i o n increases and photosynthesis becomes rate l i m i t i n g . Ormrod et a l . (1968) studied the e f f e c t of temperature on net as s i m i l a t i o n of twelve barley v a r i e t i e s and found that the e f f i c i e n c y of a plant changes as i t develops.' Azmi (1969) reported highest values of rates of net photosynthesis on a lea f blade weight basis f o r several r i c e v a r i e t i e s f o r two week old plants and the lowest rates for eight week old plants. Forsyth and H a l l (1965) observed higher rates of apparent photosynthesis i n young or middle aged leaves of lowbush blueberry than i n the older ones. 53 Adaptation to Temperature The observed rate of photosynthesis i n a plant depends not only on the momentarily p r e v a i l i n g temperature, but also on how long the plant has been exposed to i t . The exact b i o k i n e t i c range of photosynthesis depends on the i n d i v i d u a l adaptation of the plant as well as on the phylogenetic adaptation of the species. Cold adapted plants often show a maximum of net photosynthesis at temperatures below 10°C, while the high temperature adapted plants do not reach the maximum rate of net photosynthesis before 40°C, are capable of organic synthesis even at 50°C, and survive without permanent injury at 8 0°C or even 9 0°C (Rabinowitch, 1956). Inman (1940) studied the photosynthesis of thermal algae from Yellowstone National Park and found that t h i s algae normally grew at 65°C but could r e t a i n the power to carry on photosynthesis even at 20°C. Hesketh (1968) observed that f o r some species such as Helianthus annus, Phaseolus v u l g a r i s , Triticum aestivum and Amaranthus palmer, photosynthesis could change with the change i n external conditions such as temperature and l i g h t over a wide range. These species were able to grow i n a range of 15/10 to 36/31°C (day/night). However, Gossypium hirsutum and Zea mays, which were also tested, were unable to withstand the lower temperature l i m i t s . Treharne et a l . (1968) studied the growth response of orchard grass and found that when grown at 29/21°C (day/night) and measured at 54 29 and 21 C the photosynthetic rate was much higher at the temperature at which the leaves developed and grew (29°C) than when measured at a lower temperature of 21°C. M i l l e r (1960) compared the temperature e f f e c t on the rate of apparent photosynthesis i n Seaside bent and Bermuda grass and found a maximum rate for the former at 2 5°C and the l a t t e r at 3 5°C. Ormrod et a l . (1968) observed v a r i e t a l differences i n terms of net C0 9 exchange rates i n barley when measured at d i f f e r e n t temperatures and suggested that these could be r e l a t e d to o r i g i n and production areas of these v a r i e t i e s . Mooney and West (1964) i n t h e i r photosynthetic acclimation study of plants of diverse o r i g i n s , observed the p l a s t i c i t y of a number of t e r r e s t r i a l higher plant species. They were acclimated for over three weeks i n a desert and subalpine environment and t h e i r subsequent photosynthetic rates were recorded over a range of temperatures. Both rate modifications and s h i f t s i n temperatures of optimal photo-synthesis were observed. Further they found that plants of a l l species acclimated i n the coldest environment were more e f f i c i e n t i n photosynthetic a c t i v i t y at colder temperatures, and plants acclimated i n the desert environment were more e f f i c i e n t at warmer temperatures. 55 MATERIALS AND METHODS The following experiments were conducted from A p r i l 1967 to September 1969. Experiments 1, 2,4 and 6 were harvested at four weeks and experiments 3 and 5 at maturity. 1. An experiment to investigate the response of barley, peas and rape to six l e v e l s of S at two temperature regimes; l e v e l s tested were 0, 1, 4, 8, 16 and 64 ppm at 18/10 and 29/21°C (day/night). Subsequent experiments were conducted at 0 and 6 4 ppm or at 0, 8 and 64 ppm at various temperature regimes as indicated below. 2. E f f e c t of two extreme temperature regimes, low 13/4 and high 35/27°C on the growth and development of barley,, peas and rape at three S l e v e l s 0, 8 and 64 ppm at 4 weeks (vegetative stage). 3. E f f e c t of two extreme temperature regimes, low 13/4 and high 3 5/27°C and three S l e v e l s 0, 8 and 64 ppm on the growth and development and the uptake and d i s t r i b u t i o n of S and N between the shoots and f r u i t s of barley, peas and rape at maturity. 4. E f f e c t of three intermediate temperature regimes 18/10, 24/16 and 29/21°C and two S l e v e l s 0 and 64 ppm on the growth and development of barley, peas and rape at 4 weeks (vegetative stage). 56 5. E f f e c t of three intermediate temperature regimes 18/10, 24/16 and 29/21°C and three S l e v e l s 0, 8 and 64 ppm on the growth and development and the uptake and d i s t r i b u t i o n of S and N i n the shoots of barley, peas and rape at maturity. 6. E f f e c t of the removal of cotyledons or endosperm and S l e v e l s 0, 8 and 64 ppm on the growth and development of barley, peas and rape at two weeks. 7. An experiment to study the rate of net photosynthesis i n barley, peas and rape at two stages of growth; 20 and 30 days from sowing and S l e v e l s 0 and 64 ppm and four temperature regimes; 18/10, 24/16, 29/21 and 35/27°C. Photosynthetic rates were measured at the growing temperature, two 5.5°C temperature i n t e r v a l s below and two 5.5°C i n t e r v a l s above the growing day temperature of each regime. 8. E f f e c t s of temperature regimes 18/10, 24/16 and 29/21°C and S l e v e l s 0 and 64 ppm on the CC^ compensation points i n barley, peas and rape. Plant Species Three species of plants; barley, peas and rape, representing the families Gramineae, Leguminoceae and Crucifereae, r e s p e c t i v e l y , were used i n a l l the experiments. Barley (Hordeum vulgare cv O l l i ) seeds were obtained from the Research Station, Beaverlodge, Alberta; pea (Pisum sativum cv Dark Skin Perfection) seeds from the Research 57 Station, Agassiz, B r i t i s h Columbia; and rape (Brassica  campestris cv Arlo) seeds from the Research Station, Saskatoon, Saskatchewan. Growth Chambers Plants were grown i n three P e r c i v a l growth chambers (model PGC-78). Each chamber had a work area of 11 sq. f t . (1.30 sq. m.) and a growth height of 52" (1.32 m). Light was provided by a combination of 16 cool white florescent lamps and 10 25-watt incandescent bulbs per chamber. An i n t e n s i t y of 1800 to 1900 footcandles (19.4 to 2 0.5 klux) was maintained at the surface of the plants by moving the holding racks cl o s e r to or away from the l i g h t source depending on the height of the plants. Light i n t e n s i t y was measured with a Weston Model 756 Illumination Meter without cosine f i l t e r . Light was well d i s t r i b u t e d i n the chambers due to the r e f l e c t i v e white enamel walls and there was l i t t l e or no mutual shading u n t i l the plants were grown for at least 4 weeks. The photoperiod, c o n t r o l l e d by an "Intermatic" time switch, was 16 hours i n a 24 hour c y c l e , and day and night temperatures were programmed by a "Paragon" time switch to coincide with the photoperiod. The following temperature regimes were used for the various experiments 13/4, 18/10, 24/16, 29/21 and 35/27°C. These were maintained within ±0.5°C. An a i r v e l o c i t y of less than 75 fpm was maintained i n each chamber. 58 Cu l t u r a l Practices Seeds were washed several times with tap water and then with d i s t i l l e d water to remove any traces of S, i f present, then they were spread on paper towels to remove excess moisture before d i b b l i n g into v e r m i c u l i t e - f i l l e d pots, which were 1 to Ih l i t r e capacity (the former was used f o r experiments 1, 2, 4 and 6, and the l a t t e r for a l l the other experiments). Usually 7 seeds of barley or peas or about 20 seeds of rape per pot were dibbled 2 cm deep and the vermiculite was pressed down by hand. Immediately a f t e r planting a l l the treatments were i r r i g a t e d with d i s t i l l e d water. This was continued d a i l y f o r 4 days, a f t e r which time each treatment was applied using the appropriate nutrient s o l u t i o n . Hoagland's nutrient so l u t i o n (Hoagland and Arnon, 1950) was prepared and the magnesium sulphate content was adjusted to contain the following S l e v e l s : 0, 1, 4, 8, 16 and 64 ppm. Magnesium content was balanced by adding magnesium ch l o r i d e . These were applied at the rate of 2 00 ml per pot every second day u n t i l harvest. Additional amounts of water were provided by i r r i g a t i n g with d i s t i l l e d water e s p e c i a l l y f o r the treatments that were grown to maturity at higher temperatures. Thinning was done at the end of 7 days f o r a l l the treatments, leaving 4 uniform plants per pot. 59 Observations (a) Experiments 1 to 6 The decimal system developed by Maurer et a l . (196 5) fo r the assessment of morphological development i n peas was used for recording the nodal stages of a l l three species. These observations were taken every t h i r d day from 7 days a f t e r planting to harvest. At the time of harvest the following observations were recorded. (a) Shoot length - from base of plant to t i p of the terminal l e a f (b) Root length - from base of plant to the t i p of root (c) Number of t i l l e r s or side shoots (d) Fresh weights of shoot and roots separately (These were not taken for plants grown to maturity) (e) Dry weights of shoots and roots separately, obtained a f t e r drying i n a forced d r a f t oven at 8 0°C for 7 days (f) General observations such as the length of internodes, percentage of f e r t i l e pods and appearance of S deficiency symptoms (b) Experiment 7 (Photosynthesis) Photosynthesis was measured i n the manner described by Ormrod and Woolley (1966) and Azmi (1969). A Blue M Vapor-Temp con t r o l l e d r e l a t i v e humidity chamber (Model VP-400AT, 60 Blue M E l e c t r i c Co., Blue Island, 111. 60406) was used, maintaining a r e l a t i v e humidity of 60 per cent. This instrument was attached to an in f r a r e d gas analyzer forming a closed system. The in f r a r e d gas analyzer constantly monitored the carbon dioxide content of the a i r stream and t h i s i n turn was attached to a recorder. The l i g h t source placed over the top of the glass chamber was a bank of 6 Sylvania VHO cool white florescent tubes, giving a l i g h t i n t e n s i t y of 3,800 footcandles (41.0 klux) at the upper surface of the glass chamber and 800 foot-candles (8.6 klux) at the base of the chamber. The pots of plants were placed inside the chamber with the upper leaves about 2" from the upper surface of the chamber. The instrument was ca l i b r a t e d every day before s t a r t i n g measurements on plants and photosynthesis was measured within the range of about 3 0 ppm CO^ above and 3 0 ppm CO^ below the ambient concentration. The dry bulb temperature was changed every 30 minutes and 15 minutes was allowed f o r s t a b i l i z i n g at the new temperature a f t e r which CC^ exchange rates were measured f o r at least 15 minutes. Measurements were taken i n the following sequence; f i r s t at the temperature at which plants were grown then at two temperatures of 5.5°C i n t e r v a l s below and again at the o r i g i n a l temperature before measuring at two 5.5°C temperature i n t e r v a l s higher. For example i f plants were raised at 24/16°C (day/night), measurements 61 would be taken i n the following manner: 24.0 •*• 18.5 13.0 •*• 24.0 -*• 29 . 5 + 35°C After completing the photosynthesis measurements plants were harvested at ground l e v e l and t h e i r fresh weight (shoots), shoot length and l e a f area were recorded. Leaf area was measured by using an a i r flow planimeter (Paton Industries PTY Ltd., Dashwood Road, Beaumont, S. A u s t r a l i a ) . The area of both leaf and stems were recorded. Bulked plant material was then dried i n a forced d r a f t oven at 8 0°C f o r 7 days and dry weight was recorded. Measurement of Compensation Point For measuring the CC^ compensation points of barley, peas and rape, plants were grown at 18/10, 24/16 and 29/21°C at two l e v e l s of S 0 and 64 ppm. The second f u l l y opened leaves from the terminal bud of twenty day-old plants were used f o r t h i s experiment. The apparatus and method described by Downton and Tregunna (1968) was used f o r measuring the C0 9 compensation points. The leaves were detached and immediately recut under water, and the cut ends dipped i n a small w a t e r - f i l l e d v i a l which was lowered into the photosynthetic chamber. This chamber was placed i n a water bath i n order to be able to maintain a stable temperature i n the photosynthetic chamber. Usually the temperature was maintained at 21 ±0.5°C. The l i g h t was supplied by a General E l e c t r i c Cool Beam Lamp at approximately 2500 footcandles (27.0 klux). 62 The measurement of CC^ compensation i n "plus" treatments did not take more than 30 minutes but the "minus" treatments were slow to respond and took from 3 0 to 50 minutes. Measured l e a f samples were dried i n a forced d r a f t oven at 8 0°C f o r over 7 days and ground using a mortar and pestle before determination of t o t a l S analysis according to the Johnson and N i s h i t a (19 52) method. The c o r r e l a t i o n of S l e v e l s and temperature with compensation points was then ca l c u l a t e d . Chemical Analysis Chemical analysis was c a r r i e d out as follows f o r a l l the experiments excepting the photosynthesis experiment. Preparation of Material for Chemical Analysis A f t e r drying the shoots i n a forced d r a f t oven at 8 0°C f o r 7 days or more, plant material was ground i n a Wiley M i l l (intermediate model) using a 60 mesh screen u n t i l a l l the material was uniformly ground. When the quantity of plant material was large and fibrous mainly from experiments grown to maturity, samples were ground i n a larger Wiley M i l l (Model No. 3) using h nun mesh screen and reground i n the intermediate Wiley M i l l . The ground material was stored i n clean bottles or white paper envelopes and again oven dried f o r a minimum period of 12 hours p r i o r to weighing for chemical ana l y s i s . 63 The following chemical analyses were c a r r i e d out for each experiment. 1. Experiment 1 N i l 2. Experiment 2 (Shoots) Sulphate sulphur and t o t a l nitrogen 3. Experiment 3 (Shoots) Sulphate sulphur and t o t a l nitrogen 4. Experiment 4 (Shoots) Total sulphur, sulphate sulphur and t o t a l nitrogen 5. Experiment 5 (Shoots and F r u i t s ) Shoots - Total sulphur, sulphate sulphur, t o t a l nitrogen and n i t r a t e nitrogen F r u i t s - Sulphate sulphur and t o t a l nitrogen 6. Experiment 7 N i l 7. Experiment 8 Total sulphur i n leaves used f o r measuring compensation points Sulphate Sulphur Analysis Method Several methods for S determination have been reviewed from time to time by various authors and the generally accepted method i s the one reported by Johnson and Nish i t a (1952). It i s a very s e n s i t i v e microestimation method and can be employed to detect low l e v e l s of S, as low as 0.5 ppm i n plant material 64 and 0.1 ppm soluble sulphate i n s o i l s . It i s rapid and accurate and i s employed f o r determining both t o t a l sulphur and sulphate sulphur. Johnson and N i s h i t a (19 52) employed the reducing properties of hydriodic acid f o r the quantitative determination of sulphate. Sulphate was reduced to sulphide by b o i l i n g with a mixture of hydriodic acid, formic acid and red phosphorus. However, more recently, t h i s was modified and the reducing mixture used was hydriodic a c i d , formic acid and hypophosphorus aci d (Chapman and Pratt, 1961). The colour methylene blue (MB) was developed by mixing the solution containing hydrogen sulphide with an acid solution of p-aminodimethylaniline and f e r r i c i r o n . Methods of reduction and colour reaction have been c r i t i c a l l y reviewed by Gustafsson (1960 a, b), and more recently reviewed by Beaton et a l . (1968). The d i g e s t i o n - d i s t i l l a t i o n apparatus ( f i g . 1) consisted of a b o i l i n g f l a s k , water jacketed condenser, connecting U-tube, gas washing column and detachable connecting tube leading to a 100 ml volumetric f l a s k . A gas-washing bo t t l e was placed between the nitrogen tank and the i n l e t arm of the b o i l i n g f l a s k . Nitrogen was washed by bubbling i t . through a solution of mercuric chloride i n 2% potassium permanganate to remove possible traces of sulphur gases. Six of these units were placed i n a series as shown i n figure 2 with each round bottomed f l a s k r e s t i n g on a small e l e c t r i c hot plate (micro k j e l d a h l type). Ten ml of the p y r o g a l l o l 65 I 5 c m F i g . 1. Apparatus f o r the reduction of sulphate (Gustafsson, 1960b). (a) 50 ml reduction f l a s k (b) gas i n l e t tube (side arm) (c) water jacketed condenser (d) gas-washing column (e) detachable connecting tube (f) connecting U-tube 66 F i g . 2. The 6 unit apparatus used for microestimation of sulphur 67 sodium phosphate reagent was placed i n the gas-washing column and 10 ml of zinc acetate and sodium acetate solution added to 7 0 ml of sulphur-free d i s t i l l e d water i n a 100 ml glass-stoppered volumetric f l a s k . The small connecting tube was attached to the side arm of the gas washing column before placing the receiving f l a s k . Plant material containing not more than 100 mg S was introduced to the digestion f l a s k . For t o t a l sulphur analysis, 2 ml of an aliquot of the extract obtained by wet ashing was used. Four ml of reducing mixture was then added and the fl a s k quickly attached to the condenser, the side arm to the tube leading from the nitrogen wash b o t t l e . A flow of nitrogen at a rate of 150 to 200 ml per minute was maintained throughout the digestion process. Digestion was c a r r i e d out for at l e a s t one hour. At the end of t h i s period 10 ml of p-aminodimethylaniline solution was added to the receiving f l a s k and gently shaken before the addition of 2 ml of f e r r i c ammonium sulphate sol u t i o n . The f l a s k was stoppered, mixed, made to volume and mixed again. Samples were read i n a Beckman DU Spectrophotometer Model 2400 a f t e r a minimum of 20 minutes at 670 my using a s l i t width of .04 mm and 1 cm c e l l length. Total Sulphur Total sulphur was determined i n the same manner a f t e r oxidation according to the following procedure (Johnson and Ni s h i t a , 1952). Samples containing less than 100 mg S were transferred to 30 ml micro k j e l d a h l f l a s k s , 2 ml concentrated 68 n i t r i c acid added to each f l a s k , digested on a steam bath for 30-40 minutes and 1 ml of 60 percent perchloric acid added. These fla s k s were then placed on k j e l d a h l microburners and the digestion continued for 1 hour with gradually increasing heat. They were then allowed to cool and 3 ml of 6 N hydro-c h l o r i c acid added, heated again for Ih hours, cooled and made to 50 ml i n volumetric f l a s k s . Two ml aliquots were taken for determination of sulphate sulphur according to the method described i n the preceding section. Total Nitrogen and Nitrate Nitrogen Analysis for t o t a l nitrogen was done according to the semi-micro k j e l d a h l method and n i t r a t e nitrogen by a method reported by Miiller and Widemann (196 5) and modified by Ormrod and Derics (unpublished). It was further modified f o r the present analysis by using activated charcoal as a decolourizing agent instead of a copper sulphate and magnesium carbonate-calcium hydroxide mixture. 250 to 500 mg of dry plant material was placed i n a 250 ml beaker with 80 ml of water. This was heated on a steam bath for 1 hour with occasionally s t i r r i n g , f i l t e r e d and made to volume i n a 100 ml f l a s k . About 25 ml of t h i s was combined with 25 mg activated charcoal to remove any colours present, f i l t e r e d again and a 25 ml aliquot removed to which 1 ml of 0.5 percent sodium s a l i c y l a t e was added and evaporated to dryness on a steam bath. One ml of HoS0,, was added and the mixture allowed 69 to stand f o r 10 minutes. Then 6 ml of d i s t i l l e d water was added, the mixture cooled and made a l k a l i n e by adding 7 ml of 30 percent NaOH. A yellow colour indicated the presence of N0 3. Readings were made at 420 m i n a Beckman DU spectrophotometer Model 2400. • Experimental Design and S t a t i s t i c a l Analysis Experiments 1, 6, 7 and 8 were analyzed as 3 x 3 f a c t o r i a l experiments arranged i n 3 complete blocks or rows (App. 1), and each experiment was conducted only once. Each row contained the random arrangement of the 9 combinations of 3 species and 3 l e v e l s . Experiments 2, 3,4 and 5 were conducted twice and were treated as two runs i n the s t a t i s t i c a l analysis (App. 2). In experiment 3, only two re p l i c a t e s were taken from each run thus making a t o t a l of 4 r e p l i c a t e s (pots). Experiments 2, 4 and 5 had 6 r e p l i c a t e s including both runs. A l l the data were analyzed using an IBM 360 Model 6 7 Computer, Operating Michigan Terminal System (MTS). Duncan's multiple range tests (Duncan, 1955) were carri e d out i n order to determine the s i g n i f i c a n t differences among i n d i v i d u a l means. Multiple regression analysis was done on data from experiment 8 and a table of r values was prepared. 70 RESULTS General Observations Sulphur deficiency symptoms were observed i n a l l three species that were grown under S-deficient conditions. Some of the common symptoms observed were marked chl o r o s i s that was usually i n t e r v e i n a l together with r e s t r i c t i o n of shoot growth. Figures 3a, b and c show the e f f e c t of S deficiency on shoot growth of barley, peas and rape plants at a temperature regime of 18/10°C (day/night). Sulphur deficiency symptoms i n rape plants were f i r s t observed i n the older leaves and gradually spread to the younger leaves. In pea plants they f i r s t appeared i n younger leaves, and gradually spread to the older leaves, while i n barley plants the symptoms tended to appear f i r s t i n the younger leaves and spread f a i r l y soon to the older leaves. Of the three species, c h l o r o t i c symptoms were f i r s t observed i n barley followed by rape and peas. Usually these symptoms begin to appear a f t e r two weeks from planting. The lowest S l e v e l treatment at the highest temperature regime took the shortest time to develop deficiency symptoms. With increase i n S le v e l s or decrease i n temperature or both, development of deficiency symptoms was delayed or the symptoms did not appear at a l l . With adequate supply of S, a l l three species grew well at temperature regimes that were not too E f f e c t of sulphur l e v e l s (0, 8 and 64 ppm) on growth of barley and peas at 18/10°C (day/night). Ten weeks from sowing. F i g . 3c. E f f e c t of sulphur lev e l s (0, 8 and 64 ppm) on the growth of rape at 18/10°C (day/ night). Ten weeks from sowing. 73 low or too high. Plant weight, plant height, number of t i l l e r s or shoots and the number of f e r t i l e f r u i t s increased. Dark green colour of the leaves was also a common feature of these plants (Figures 3a, b and c ) . Generally, a l l three species grown at low temperature regimes had an i n i t i a l delay i n growth, mainly due to the long time required f o r germination. For example, rape seeds sown i n the 35/27°C growth chamber, germinated i n 2 days compared to the seeds sown i n the 13/4°C growth chamber that germinated a f t e r 7 days. E f f e c t of Sulphur N u t r i t i o n on Growth, Mineral Concentration  and Total Uptake i n the Shoots at M- Weeks (Vegetative Stage)  as Influenced by Temperature Combined analysis of variance with the three species was c a r r i e d out. F i r s t order interactions between temperature and S, temperature and species, and species and S l e v e l were observed. Second order interactions between temperature, species and S l e v e l s were also evident as indicated i n table 1. Simple e f f e c t s of S at each temperature regime f o r each species i 1 were therefore determined i n subsequent analyses. Growth With an increase i n S l e v e l there was an increase i n both fresh and dry weights of barley plants (Tables 2 and 3). These weight increases were not s i g n i f i c a n t l y d i f f e r e n t at 18/10°C. Shoot weights increased with increasing sulphur Table 1. F values and the significances of main effects and interactions for each measurement at 4 weeks (vegetative stage) Treat-ment e f f e c t s Temper-ature Species Level SxL TxS TxL TxSxL Shoot Root Total Shoot fresh fresh fresh dry weight weight weight weight Root Total dry dry weight weight Percent of dry matter (shoot) Total uptake per shoot Nodes N SO, -S N 62.38** 11.02* 38.21** 154.59** 51.46** 144.46** 10.96** 440.29** 58.31** 15.60** 134.78** 43.46** 18.79** 5.43** 40.00** 7.97** 6.32** 8.28** 16.30** 11.45** 4.35** 3. 80*' 10.655 4.35* 31.90** 20.33** 7.96** 6.44** 6.81** 9.6 0** 7.55** 3.0 3* 7.59** 10.46** 4.30** 3.72* 9.27** 16.95** 7.88** 6.81* 7.52** 4.06** 2016.13** 12.33* 76.00** 428.31** 91.34** 33.19** 33.33** 146.35** 10.87** 6.27** 30.29** 3.97** 232.31** 2.92ns 13.62** 13.40** 0.41ns 1.50ns 7.30** 1.33ns 1.65ns 90.40** 49.80** 72.46** 20.92** 277.41** 17.62** 36.49** 8.89** 43.07** 16.63** 70.67** 6.82** 46.77** 4.24** ns Non s i g n i f i c a n t * S i g n i f i c a n t at P = 0.0 5 ** S i g n i f i c a n t at P = 0.01 Table 2. E f f e c t of temperature and sulphur n u t r i t i o n on the growth of barley, peas and rape plants at 4 weeks (vegetative stage). Experiment 1 Temperature Sulphur Shoot fresh Root fresh Total fresh Shoot dry Root dry Total dry day/night l e v e l weight weight weight weight weight weight °C (ppm) g g g g g g Nodes 18/10 0 2.98a 1.47a 4.44a 0 .35a 0.16a 0.51a 4.3b 1 3.20a 1.49a 4.69a 0.37a 0.16a 0 .53a 4.6b 4 3.19a 1.88a 5.07a 0.3 8a 0.13a 0.51a 4.6b 8 4.08a 1.67a 5.75a 0.41a 0.14a 0.55a 5.1a 16 4.45a 1.95a 6 .40a 0.43a 0.13a 0.57a 5 . 0a 64 4.51a 1.97a 6.48a 0.46a 0.15a 0.62a 5.2a 29/21 0 1.82b 1.19b 3.01b 0.24b 0.11b 0.35b 5.1c 1 2.42b 1.63b 4.04b 0.31b 0.13b 0.45b 5.2c 4 4.41a 2.58a 6.99a 0.60a 0.28a 0.88a 5.8b 8 4.58a 1.99b 6.57a 0.59a 0.30a 0.81a 5. 8b 16 4.04a 3 ,09a 7 .13a 0.60a 0.30a 0.90a 6 . Oab 64 4.48a 2.38a 6.87a 0 .65a 0 .30a 0.96a 6.4a 18/10 0 3.44a 2.74b 6.18b 0.33b 0 .23c 0.55c 4.5c 1 3.57a 2.82b 6.38b 0.36b 0 .28bc 0.36bc 4 .5c 4 3.61a 4.26a 7.87a 0.37b 0 .33a 0.69b 4.9bc 8 3.63a 4.13a 7.76a 0.37b 0.33a 0.70b 5 . 6ab 16 3.64a 4.78a 8.42a 0.39b 0.32ab 0.71ab 5. 6ab 64 4.03a 4.38a 8.41a 0.49a 0.35a 0.84a 5 .8a 29/21 0 3.42d 4.32a 7.74b 0.49d 0.19d 0 .69d 9. Id 1 3.60cd 4.37a 7.97b 0.54cd 0 .30c 0.84cd 9.5cd 4 4.37bc 4.19a 8.56b 0.62bc 0 .31bc 0 ,93bc 9 . 8bcd 8 4.60b 4.09a 8.69b 0.65b 0.34bc 0.99b lO.Oabc 16 4.77ab 4.16a 8.92b 0.68b 0.35ab 1.03b 10.5ab 64 6.23a 4.17a 10.40a 0.91a 0.40a 1.31a 10.7a Table 2. (continued) Temperature Sulphur Shoot fresh Root fresh Total fresh Shoot dry Root dry Total dry day/night l e v e l weight weight weight weight weight weight Species °C (ppm) g g g g g g Nodes Rape 18/10 29/21 0 1.23b 0.23a 1.46b 0.14b 0.02a 0.16b 4.4a 1 4.11a 0.47a 4.58a 0.33a 0.04a 0 .37ab 4.1a 4 4.17a 0.44a 4.61a 0.33a 0.04a 0.37ab 4.1a 8 4.15a 0.47a 4.62a 0.35a 0 .05a 0.40ab 4.6a 16 4.18a 0.77a 4.99a 0 .36a 0.06a 0.42ab 4.6a 64 5.03a 0.55a 5.57a 0.44a 0 .04a 0.48a 4.7a 0 3.15d 0.69b 3.84c 0.35c 0.07c 0.42c 6.1b 1 3.00d 0.82b 3 . 82c 0.30bc 0.08c 0.38bc 6.2b 4 4.22cd 1.15b 5.37c 0.39abc 0.10c 0 .49abc 6 .8b 8 4.97bc 1.25b 6.25bc 0.43abc 0.08c 0.50abc 6.8b 16 6.52b 1.48b 7.99ab 0.60ab 0.15b 0.75ab 6.9b 64 8.84a 3.25a 12.09a 0.83a 0.40a 1.23a 9.7a * Each figure i s the mean of 3 r e p l i c a t e s . Figures followed by the same l e t t e r within a species, temperature regime and measurement are not s i g n i f i c a n t l y d i f f e r e n t at P = 0.05 according to Duncan's new multiple range t e s t . CD Table 3. Growth c h a r a c t e r i s t i c s and y i e l d factors i n barley, peas and rape as influenced by temperature and sulphur n u t r i t i o n at 4 weeks (vegetative stage). Experiment 4 Species Barley Peas Rape Temperature Sulphur Shoot fresh Root fresh Total fresh Shoot dry Root dry To t a l c day/night l e v e l weight weight weight weight weight weighl °C (ppm) g g g g g g 18/10 0 A 0* 4.49b 1.29a 5.78a 0.64a 0.16a 0.79a 64 6.70a 1.44a 8.14a 0.81a 0.17a 0.9 8a 24/16 0 4.66b 1.81b 6.47b 0.65b 0.19a 0.83b 64 11.40a 3. 88a 15.28a 1.17a 0.23a 1.39a 29/21 0 2.26a 0 .85a 3.11a 0.39a 0.11a 0.50a 64 3 .85a 1.37a 5.22a 0.62a 0.18a 0.79a 18/10 0 4.58b 3.7 8a 8. 36a 0.62b 0.29a 0.92b 64 6.03a 3.22a 9.25a 0.77a 0.27a 1.05a 24/16 0 4.94b 4.99b 9.94b 0.61b 0.38a 0.99b 64 8.65a 6.69a 15.57a 0.96a 0 .39a 1.35a 29/21 0 4.66b 3 ,39a 8.00a 0.67b 0.28a 0.96b 64 5 .95a 3 .03a 8.98a 0. 89a 0.29a 1.19a 18/10 0 0.84b 0.65b 1.49b 0.10b 0.07a 0.17b 64 4.30a 2.50a 6 .80a 0.43a - 0.20a 0.63a 24/16 0 0.73b 0 .55a 1.28b 0.13b 0 .05a 0.18a 64 4.49a 1.43a 5.92a 0.45a 0.15a 0.59a 29/21 0 0.79b 0.51b 1.30b 0.14b 0.05b 0.19b 64 9.09a 4.18a 13.28a 0.93a 0.56a 1.50a Each figure i s the mean of 6 r e p l i c a t e s . Figures followed by the same l e t t e r within a species, temperature regime and a measurement are not s i g n i f i c a n t l y d i f f e r e n t at P = 0.05 according to Duncan's new multiple range t e s t . 78 more than root weights. However, at 29/21°C, the two lowest l e v e l s of S caused s i g n i f i c a n t l y lower plant weights than the higher l e v e l s . Generally i t was evident that barley plants responded better to S at 24/16 than at 18/10 or 29/21°C (Table 3). Similar to barley, the shoot weights and root weights of pea plants were also increased at 24/16 and 29/21°C with increase i n the rate of S (Table 3). Generally the best response to S was observed at 24/16°C as shown i n table 3. Highest values f o r rape plants grown with or without S were obtained at the higher temperature regimes p a r t i c u l a r l y at 29/21°C. Nodes Number of nodes of a l l three species were affected by both temperature and S l e v e l s (Table 2). Plants grown at lower temperature regimes with lower l e v e l s of S had fewer nodes compared to those grown at higher temperatures with higher l e v e l s of S. S i g n i f i c a n t differences among l e v e l s within temperatures were found i n barley and peas at both 18/10 and 29/21°C and in-rape only at 29/21°C between 64 ppm and a l l the other l e v e l s . Mineral Concentration and Total Uptake  Total Sulphur Sulphur concentration was expressed as percent of shoot dry matter (Table 4). A l l three species had s i g n i f i c a n t l y higher S concentration in the tissue when grown with 64 ppm S Table 4. E f f e c t of temperature and sulphur n u t r i t i o n on the mineral concentration and t o t a l uptake per plant shoot, i n barley, peas and rape at 4 weeks (vegetative stage). Experiment 4 Species Temperature day/night °C Sulphur l e v e l <PPm> Percent of dry matter so 4 =s N Total uptake (mg) S SO.-S N N/S r a t i o Barley Peas Rape 18/10 0 0, .040b 0 .011b 3. .04b 0. ,25b 0. ,07b 19, ,5b 76. . 0a 64 0, .322a 0 .148a 5, .06a 2, ,73a 1. ,09a 42, .7a 15. ,7b 24/16 0 0, .071b 0 • 007b 3 . ,00b 0, ,44b 0, ,03b 23, ,5b 42, ,2a 64 0, .307a 0 .125a 5, ,05a 3, . 60a 1, .44a 59, ,2a 16. ,4b 29/21 0 0, .159b 0 ,008b 3, .05b 0, ,5 8b 0, ,03b 12, . 0b 19, ,1a 64 0, .3 81a 0 .125a 3, . 85a 2, , 36a 0, ,73a 47, . 3a 10, .lb 18/10 0 0. .108b 0 .008b 4. ,10a 0. ,54b 0. ,04b 25. ,5b 37. . 9a 64 0, .314a 0 .102a 4. ,66a 2. ,41a 0. ,73a 36, ,4a 14. ,8b 24/16 0 0, .133b 0 .006b 4, ,49a 0, . 53a 0, ,02b 30, .2b 34. , 5a 64 0, .272a 0 .114a 4, ,46a 2, ,59a 1. . 09a 52, . 3a 16, ,3b 29/21 0 0, .225b 0 .011b 4. ,75a 0, .87b 0, ,07b 32, ,0b 21. ,1a 64 0. ,369a 0 .193a 4. ,81a 3, .2 8a 1. , 71a 42, . 8a 13, .0b 18/10 0 0, .068b 0 .006b 4, ,48a 0, .05b 0. ,01b 4, ,5b 65 , . 9a 64 0, .799a 0 .495a 3, ,84a 3, .43a 2, ,15a 16, .7a 4, ,8b 24/16 0 0, .068b 0 . 005b 3, ,00b 0, .05b 0, ,01b 4, ,0a 44, ,1a 64 0, .877a 0 .506a 3, . 65a 4, .85a 2, ,64a 20, ,4a 4, ,1b 29/21 0 0, .053b 0 .008b 3, ,10a 0, .06b 0, ,01b 4, .2b 58, . 5a 64 0, .737a 0 .557a 3. ,50a 6, . 65a 4, ,29a 31, .4a 4. .7b Each figure i s the mean of 6 r e p l i c a t e s . Figures followed by the same l e t t e r within each species, temperature regime and a p a r t i c u l a r element are not s i g n i f i c a n t l y d i f f e r e n t at P = 0.05 according to Duncan's new multiple range t e s t . t o 80 than with 0 ppm S. Total S content of barley and peas grown at the low S l e v e l was increased as the temperature regime was raise d , p a r t i c u l a r l y when the temperature was increased from 24/16 to 29/21°C. This increase i n concentration was not observed i n rape. The e f f e c t of temperature on the t o t a l uptake of S per plant at the 0 ppm l e v e l was s i m i l a r to the e f f e c t on S concentration, but at 64 ppm the t o t a l uptake had a d i f f e r e n t trend. At t h i s l e v e l rape and peas had higher t o t a l uptake with increase i n temperature, whereas the t o t a l uptake i n barley increased at 24/16 and decreased at 29/21°C. Of the three species rape had the highest S uptake at a l l temperature regimes. Barley had a higher uptake than peas at 24/16 but peas had a higher uptake than barley at 29/21°C (Table 4). Sulphate Sulphur Concentration and Total Uptake Percent SO^-S i n 4 week old plant shoots of the three species grown at 4 temperature regimes are given i n tables 4 and 5. S i g n i f i c a n t differences between SO^TS concentration within temperature regimes were present f o r d i f f e r e n t S l e v e l s . Greatest concentration was at the highest l e v e l (64 ppm) and lowest concentration at the lowest l e v e l (0 ppm). Plants grown at 35/27°C had higher concentrations of SO^-S than those grown at 13/4°C i n a l l three species. This difference was greatest i n rape. Data i n table 4 also show the increase i n SO,,-S with increase i n 81 Table 5. E f f e c t of temperature and sulphur n u t r i t i o n on the concentration and t o t a l uptake of sulphate sulphur and nitrogen i n the shoots of barley, peas and rape at 4 weeks (vegetative stage). Experiment 2 Total uptake m a. o -, , Percent of per plant Temperature Sulphur , . . -u l. / day/night l e v e l dry matter shoot/mg Species °C (ppm) SO^-S N SC^-S N Barley Peas Rape 13/4 0 0.015b 4 .40b 0 .043c 12 .00b 8 0.057b 4 .53b 0.191b 15 .50b 64 0.179a 5 .30a 0.818a 24 .02a 35/27 0 0.013b 3 . 36b 0.131b 24 .55b 8 0.053b 3 . 60ab 0.507b 28 . 3 3ab 64 0.209a 3 .9 0a 1. 845a 33 .82a 13/4 0 0.040a 5 .27a 0.163a 21 .05a 8 0.122a 5 • 27a 0.507a 21 .85a 64 0.213a 5 .25a 0.937a 22 .77a 35/27 0 0.025b 5 .75a 0.297b 63 .75a 8 0.130b 5 .52a 1.513b 68 .80a 64 0.324a 5 .44a 4.578a 42 .43a 13/4 0 0.035b 4 . 83a 0.050a 6 .92a 8 0.163ab 4 .95a 0.260a 7 .32a 64 0.288a 5 .54a 0 . 503a 9 • 10a 35/27 0 0.16c 3 .52b 0.121c 25 .65b 8 0.201b 4 .20b 2.323b 55 .13a 64 0,840a 5 .07a 11.015a 71 .32a * Each figure i s the mean of 6 r e p l i c a t e s . Figures followed by the same l e t t e r within a species, temperature regime and a p a r t i c u l a r element are not s i g n i f i c a n t l y d i f f e r e n t at P = 0.05 according to Duncan's new multiple range t e s t . 82 temperature for peas and rape. Barley, on the other hand, had the greatest increase at 18/10°C and s l i g h t l y smaller increases at 24/16 and 29/21°C at both l e v e l s . Similar to SO^-S concentration, s i g n i f i c a n t differences between le v e l s within temperatures were observed i n t o t a l uptake of SO^-S i n the shoots of the three species. Generally uptake was increased at higher temperatures and the greatest increase was found i n rape at 35/27°C as i s apparent from table 5. Nitrogen Concentration and Total Uptake Total N concentration was found to be s i g n i f i c a n t l y greater at higher S l e v e l s than at lower S l e v e l s i n barley and rape. There were no s i g n i f i c a n t differences i n N concentration i n peas (Tables 4 and 5). N concentration was high at 13/4 and 35/27°C f o r the highest S l e v e l i n rape and peas, whereas barley from 64 and 8 ppm S l e v e l s had the highest N concentration at 18/10 and lowest concentration at 35/27°C. Barley and rape plants grown with the lowest S l e v e l had the highest concentration of N at the lowest temperature regimes and pea plants tended to have the highest concentration of N at both extreme temperatures. Total uptake of N was s i g n i f i c a n t l y d i f f e r e n t between le v e l s i n a l l three species at a l l temperature regimes. Generally the amount taken up depended on the amount of S supplied to the plant as seen i n table 5. At 64 ppm, barley and peas had the greatest N uptake at 24/16°C and t o t a l uptake 83 decreased with increase i n temperature (Table 4). On the other hand, the amount taken up by rape increased with increase i n temperature. Nitrogen/Sulphur Ratio Greater N:S r a t i o s for S-deficient plants were found as shown i n table 4. These values were s i g n i f i c a n t l y greater than values obtained for plants grown with adequate S. Greatest difference between r a t i o s f o r the two l e v e l s i n barley was present at 18/10 and for peas at 24/16°C. Ratio differences f o r rape at the three temperature regimes were about the same. E f f e c t of Temperature and Sulphur N u t r i t i o n on the Growth,  Mineral Concentration and Total Mineral Uptake i n Barley,  Peas and Rape Plants at Mature Stage  Growth As i s evident from tables 6 and 7 dry weights of shoot and root at 0 ppm S l e v e l were s i g n i f i c a n t l y lower than at the 8 or 64 ppm S leve l s at a l l temperature regimes excepting i n barley and peas at 35/27°C. Greatest shoot and root weights f o r barley and peas were present at 13/4 and 18/10°C temperature regimes at a l l three S l e v e l s . Decrease i n weight due to high temperature was greater for barley than f o r peas. E f f e c t of S on rape plants was more pronounced at 29/21°C than at the other temperature regimes. Overall increases i n dry wegihts were observed at both extreme temperatures (13/4 and 35/27°C) i n rape plants. Table 6. E f f e c t of temperature and sulphur n u t r i t i o n on the growth of barley, peas and rape at mature stage. Experiment 5 Species Barley Peas Rape Temperature Sulphur Shoot dry Root dry Shoot Root day/night l e v e l weight weight length length °C (ppm) g g cm cm Nodes 18/10 0 0 .73b 0 .24b 48 .76b 33 .7 8b 6 . 5a 8 11 .14a 2 .16a 90 .42a 38 .60a 6 .0b 64 10 .40a 2 .01a 95 .75a 40 . 89a 6 ,0b 24/16 0 0 .76b 0 .22b 50 .03b 30 .98a 6 .9a 8 6 .72a 1 • 79a 90 .17a 29 .46a 6 .5b 64 6 . 35a 1 .43a 78 .7 4a 24 .13b 6 .6b 29/21 0 0 . 83b 0 • 2 3b 54 .10b 25 .90a 6 .7a 8 4 .00a 0 . 87a 70 .35a 22 .35b 6 .7a 64 4 .74a 0 .97a 66 . 80a 23 . 36ab 6 .7a 18/10 0 1 .37b 0 .53c 49 .53b 38 . 35a 15 .2b 8 13 . 35a 1 .67a 97 .28a 36 .06b 18 . l a 64 12 .15a 1 . 35b 93 • 21a 34 .03b 18 . l a 24/16 0 1 .30b 0 .49b 48 .0 0b 29 .46a 16 . 6a 8 5 . 65a 0 .7 7a 73 • 91a 27 .17b 16 .4a 64 5 .54a 0 .76a 78 .48a 25 .65b 17 .4a 29/21 0 1 .08b 0 ,41b 41 .65b 27 .68a 17 .3b 8 2 .90a 0 .5 2a 55 • 12a 24 .89b 18 . 3ab 64 3 .44a 0 .73a 57 .40a 25 . 6 Sab 18 .8a 18/10 0 0 .22b 0 .11a 29 .72b 22 .6 0b 8 .3b 8 6 .13a 0 . 81a 81 .79a 34 .29a 10 . 8a 64 6 .15a 0 .73a 95 .18a 28 . 35a 11 . 6a 24/16 0 0 .37b 0 . l i b 29 .97b 21 .84a 6 .7b 8 6 .04a 1 . 53a 105 .91a 28 .19a 14 . 5a 64 5 .44a 2 .38a 95 .25a 24 .13a 14 . l a 29/21 0 0 .23b 0 .10b 12 .95b 19 .05a 6 .9b 8 6 . 5 5a 3 .14a 108 .20a 26 • 16a 14 . 8a 64 7 .37a 3 .62a 98 . 55a 24 .38a 16 . l a Each figure i s the mean of 6 r e p l i c a t e s . Figures followed by the same l e t t e r within a species, temperature regime and a p a r t i c u l a r growth measurement are not s i g n i f i c a n t l y d i f f e r e n t at P = 0.05 according to Duncan's new multiple range tes t. Table 7. E f f e c t of temperature and sulphur n u t r i t i o n on the growth of barley, peas and rape at mature stage. Experiment 3 Species Barley Peas Rape Temperature day/night °C Sulphur l e v e l (ppm) Shoot dry weight g Root dry weight g Shoot length cm Root length cm Nodes 13/4 0 1 ft .70b 0. ,40b 59, ,38b 34. ,18a 6, .la 8 10 .92a 4, .09a 88, ,69a 30, ,14b 5, . 8a 64 12 .23a 5 , .23a 88, . 03a 30, ,27b 5, . 8a 35/27 0 1 .28a 0, ,21a 36, .57a 17 , . 98a 3, .4b 8 2 .00a 0, .25a 42, .11a 14, . 90a 9, . 5a 64 3 .13a 0, , 36a 45. ,57a 15, . 31a 10, . 5a 13/4 0 1 .74b 0, ,59b 50, . 80b 39, .70a 14. .9b 8 12 . 85a 1, , 66a 94, .28a 39. ,51a 19, . 0a 64 12 . 80a 1, .92a 91. ,00a 41, ,27a 18, . 5a 35/27 0 0 .48a 0, ,09a 19. ,05b 16, ,51a 14. ,9b 8 0 .61a 0, .13a 24. .76ab 19, . 88a 16, , 5a 64 • 0 .78a 0. ,12a 26, ,77a 19, . 88a 16, . 5a 13/4 0 0 .73c 0, ,21b 48. ,56b 23, ,41a 4, .9c 8 7 .75b 1, ,02b 95. ,98a 23, ,69a 8, .0b 64 13 .3 0a 5. .03a 106. .78a 26, .44a 10, ,5a 35/27 0 3 .21b 0, .88b 32. .15b 25, .04a 1, .3b 8 12 .16a 7. ,58a 81, .68a 19, ,5 8a 22, . 5a 64 14 .14a 7. ,57a 96. .92a 20, ,44a 23, , 3a Each figu r e i s the mean of 4 r e p l i c a t e s . Figures followed by the same l e t t e r within each species, temperature regime and a p a r t i c u l a r measurement are not s i g n i f i c a n t l y d i f f e r e n t at P = 0.05 according to Duncan's new multiple range t e s t . OO cn 86 Shoot length and root length i n barley and peas decreased progressively with increase i n temperature. Shoot lengths were affected more by temperature than root lengths. This pattern was observed f o r a l l three S l e v e l s . Shoot lengths of rape plants grown with 64 ppm S were s i m i l a r at a l l temperature regimes. Root lengths on the other hand decreased s l i g h t l y with increase i n temperature up to 29/21°C and decreased considerably at 35/27°C. Plants with lowest sulphur l e v e l s had s i g n i f i c a n t l y shorter shoot lengths i n a l l three species at a l l temperatures excepting barley at 35/27°C fo r which shoot lengths were also shorter though not s i g n i f i c a n t l y so (Tables 6 and 7). Root lengths of barley were s i g n i f i c a n t l y greater at lower S l e v e l s at a l l temperature regimes excepting at 18/10°C, where the lengths were s i g n i f i c a n t l y greater at higher S l e v e l s . Root lengths of peas grown under S d e f i c i e n t conditions were s i g n i f i c a n t l y greater only at the three intermediate temperatures. Rape plants grown with adequate S had greater root lengths at a l l temperature regimes excepting at 35/27°C. 87 Nodes Number of nodes at the mature stage varied with the l e v e l of S used (Tables 6 and 7). S i g n i f i c a n t l y larger numbers of nodes were recorded i n plants grown with S of a l l three species at a l l temperature regimes, excepting i n barley at 18/10 and 24/16°C. Generally, the number of nodes also increased with increase i n temperature. Mineral Concentration and Total Uptake  Total Sulphur Concentration Total S concentrations i n the shoot tissue of barley and peas grown with 64 ppm S were s i g n i f i c a n t l y greater than those grown with 0 and 8 ppm S at 18/10 and 24/16°C (Table 8). At 29/21°C, S concentrations i n barley and peas increased s i g n i f i c a n t l y with each increase i n the amount of S supplied. S i g n i f i c a n t differences between a l l the three l e v e l s i n rape plants occurred at 24/16°C and s i g n i f i c a n t differences between the lowest and the highest l e v e l were present at 18/10 and 29/21°C. There was an increase with increased temperature i n S concentration i n the tiss u e of plants grown with adequate S. A l l three species had increased S concentration with increase i n temperature from 18/10 to 24/16°C. This increase continued even at 29/21°C f o r peas. Barley had lower S concentration i f grown at a temperature above 24/16°C. S concentration i n rape did not change by increasing temperature from 24/16 to Table 8. E f f e c t of temperature and sulphur n u t r i t i o n on the concentration of sulphur and nitrogen i n the shoots of barley, peas and rape at maturity. Experiment 5 Species Barley Temperature Sulphur day/night l e v e l Percent of dry matter Total uptake (mg) Peas Rape N/S °c (ppm) s SO, -S N N0--N S SO N NO •3^ r a t i o 18/10 0 0 .047b 0 .015b 2 .87a 0 • 207b 0 . 3 3c 0. l i b 19 . 36c 1. 41b 61 .06a 8 0 . 073b 0 .013b 1 .45c 0 . 378a 3 .88b 0. 75b 78 .30b 20. 89a 19 .86b 64 0 . 339a 0 .263a 1 . 79b 0 . 380a 18 .49a 14. 03a 96 .06a 20. 09a 5 .28c 24/16 0 0 . 050b 0 .012b 2 . 69a 0 .667a 0 .50c 0. 12b 20 .15b 4 . 9 3b 53 .80a 8 0 .118b 0 .029b 2 .24b 0 .672a 5 . l i b 1. 29b 96 .05a 28 . 2 5ab 18 .98b 64 0 .382a 0 .251a 2 .51a 0 .632a 14 .75a 9 . 69a 97 .00a 40 . 13a 6 .57c 29/21 0 0 .057c 0 .021c 2 .67a 0 .684a 0 .47c 0. 18c 22 .37c 5. 71a 47 .19a 8 0 • 181b 0 .084b 2 .31b 0 . 593a 4 . 84b 2. 52b 75 .31b 20. 49a 17 .76b 64 0 . 333a 0 . 228a 2 .57a 0 • 591a 13 .34a 9 . 20a 102 . 39a 23. 42a 7 .72c 18/10 . 0 0 .052b 0 .00 8b 4 ,64a 0 • 116a 0 .5 2b 0. 0 8b 50 . 80c 1. 23a 89 .23a 8 0 . 0 5 3 ab 0 .020b 1 .45b 0 .034a 4 .2 8b 0. 62b 114 .34a 2. 55a 27 . 35b 64 0 .112a 0 .075a 1 .42b 0 .038a 7 .16a 4. 75a 116 . 37a 2. 49a 12 .67c 24/16 0 0 . 067b 0 .006b 4 .48a 0 .222a 0 . 56c 0 . 05b 38 .0 8b 1. 82b 66 .86a 8 0 .076b 0 .018b 1 .92b 0 .095a 2 • 09b 0. 48b 52 . 5 5ab 2 . 5 6ab 25 .26b 64 0 .172a 0 • I l i a 2 .10b 0 .151a 5 .01a 3. 13a 59 .32a 4. 22a 12 .21c 29/21 0 0 .035c 0 .005c 4 .49a 0 .348a 0 .28c 0. 04b 36 .72a 2 . 8 3b 128 • 28a 8 0 .099b 0 • 037b 2 .56b 0 . 369a 1 .67b 0. 5 8b 41 ,52a 5. 35a 25 .75b 64 0 .211a 0 .151a 2 .68b 0 .248a 3 .75a 2. 69a 46 .81a 5. 60a 12 . 70c 18/10 0 0 . 083b 0 .038b 3 .78a 0 . 382a 0 .22b 0. l i b 8 . l i b 0 . 5 7b 45 .54a 8 0 • 150b 0 .091b 2 .17b 0 .414a 8 .3 7b 4. 55b 114 .15a 24. 77a 14 .47b 64 0 .609a 0 .497a 3 .52a 0 .329a 23 .71a 19. 82a 125 .82a 26 . 51a 5 .78c 24/16 0 0 . 069c 0 .027c 3 . 50a 0 .400a 0 .26b 0. 08b 12 .6 3b 2. 66c 50 ,72a 8 0 . 215b 0 . 039b 2 .13b 0 .433a 9 .42b 4 . 50b 113 .45a 23 . 80b 9 .90b 64 0 . 675a 0 .473a 2 .51b 0 . 387a 34 . 76a 24. 67a 130 .70a 46. 37a 3 .72b 29/21 0 0 .059b 0 .045b 3 .78a 0 .455a 0 .12b 0. 06b 8 .62b 1. 05b 64 .07a 8 0 .156b 0 .042b 2 .37b 0 .522a 10 .17b 4. 43b 156 .24a 34. 06a 15 .19b 64 0 .661a 0 .475a 2 .0 3b 0 . 338a 47 .62a 33. 79a 165 .35a 24. 95a 3 .07c " Each figure i s the mean of 6 r e p l i c a t e s . Figures followed by the same l e t t e r within a species, temperature regime and a p a r t i c u l a r element are not s i g n i f i c a n t l y d i f f e r e n t at P = 0.05 according to Duncan's new multiple range t e s t . 89 29/27°C. Data i n table 8 show that at the 0 ppm S l e v e l , concentrations of S i n the shoot tissue of barley and peas were s l i g h t l y increased with increased temperature excepting f o r peas at 29/21°C. Rape tissue was observed to decrease i n S concentration at the 0 ppm l e v e l with increase i n temperature. Total Uptake of Sulphur In S-deficient plants increase i n temperature from 18/10 to 24/16°C tended to s l i g h t l y increase the uptake of s u l -phur by a l l three species. Uptake was maintained at the same l e v e l at 29/21°C i n barley, and decreased i n peas and rape at the higher temperature. Barley plants grown at 8 ppm showed the same trend as d e f i c i e n t plants, whereas barley plants grown at 64 ppm S had greater uptake at 18/10°C. Total uptake i n peas was less at 24/16 than that at 18/10°C fo r both 8 and 64 ppm S l e v e l s . Rape plants on the other hand, at 8 and 64 ppm S l e v e l s , had greater uptake at higher temperature regimes (Table 8). Sulphate Sulphur Concentration S i g n i f i c a n t differences i n SO^-S between l e v e l s and between temperatures were recorded f o r the three species (Tables 8 and 9). Zero and 8 ppm S l e v e l s showed an increase i n concen-t r a t i o n with increase i n temperature for a l l three species. At the 64 ppm l e v e l , SO^-S concentration i n barley shoot tissue was s l i g h t l y lower at higher temperatures. SO^-S concentration i n peas increased progressively with temperature. Rape plants had a 90 Table 9. E f f e c t of temperature and sulphur n u t r i t i o n on the concentration of sulphate sulphur and t o t a l nitrogen i n the shoot of barley, peas and rape at mature stage. Experiment 3 Temperature Sulphur day/night l e v e l Species °C (ppm) Barley 13/4 0 0 8 0 64 0 35/27 0 0 8 0 64 0 Peas 13/4 0 0 8 0 64 0 35/27 0 0 8 0 64 0 Percent of Total uptake dry matter mg N ^ 4 N ,01b 2.82a 0.09b 32.7b .02b 1.19b 1.20b 67.1a .22a 1.39b 14.40a 88. 5a .03b 2.8 8a 0. 38a 3 6.8b . 0 8ab 2.40a 1.66a 48.1b .13a 2 . 82a 4.18a 88.9a .Ola 4. 86a 0.13b 59 .9b .02a 1.85b 1.36b 145 .9a . 06a 1.86b 4.10a 135.7a .04b 4.36a 0.18b 21.5a .10b 3 .13b 0.6 0b 19 .9a .59a 3.3 3 ab 4. 89a 26.7a Rape 13/4 35/27 0 8 64 0 8 64 0, 0, 0, 0, 0, 0, 01b 02b 31a 02b 04b 28a 2.05a 1.55a 1.79a 2 .95a 2.26ab 1.33b 0, 1, 37, 0, 4, 39, 05b 08b 40a 73b 3 6b 63a 15 112 226 99 274 183 0b 2b 6a lb 7a 9ab " Each figure i s the mean of 4 r e p l i c a t e s . Figures followed by the same l e t t e r within each species, temperature regimes and a p a r t i c u l a r measurement are not s i g n i f i c a n t l y d i f f e r e n t at P =0.05 according to Duncan's new multiple range t e s t . 91 consistently high concentration at the three intermediate temperatures (Tables 8 and 9). Total Uptake Sulphate Sulphur Total accumulation of SO^-S i n the shoots of the three species was s i g n i f i c a n t l y greater at the highest S l e v e l than at the two lower S l e v e l s at a l l temperature regimes excepting f o r barley at 35/27°C (Tables 8 and 9). At 64 ppm rape plants had more SO^-S uptake at higher temperatures, than did the other two species. On the other hand t o t a l uptake i n barley and peas tended to decrease at higher temperatures. At 8 ppm t o t a l uptake by barley increased with increase i n temperature, whereas the uptake by peas was f a i r l y constant at the intermediate temperature with the smallest uptake being at the highest temperature. At 0 ppm l e v e l , barley and peas had increased uptake at higher temperatures, while uptake by rape had no temperature e f f e c t . Total Nitrogen Concentration Nitrogen concentration was highest when the three species were grown at the 0 l e v e l of S (Tables 8 and 9). This increase i n N was s i g n i f i c a n t l y higher at the lowest S l e v e l . Barley was least affected by temperature at a l l three l e v e l s . N concentration i n peas was s l i g h t l y increased by higher temp-eratures. Temperature also had l i t t l e e f f e c t on the concentration of N i n rape. At higher S l e v e l s barley and peas had a higher concentration at 24/16 than at 18/10°C while i n rape the concentration decreased with increase i n temperature. 92 Total Nitrogen Uptake Total N uptake was s i g n i f i c a n t l y lower at the lower S l e v e l than at the two higher S l e v e l s i n a l l three species. There were no appreciable differences due to temperature at the highest and the lowest S l e v e l i n barley plants; however, the second highest l e v e l (8 ppm) had a greater increase i n N uptake at 24/16 than at 18/10 or 29/21°C. At a l l three l e v e l s , peas had less t o t a l N uptake with increase i n temperature, (Tables 8 and 9). Rape plants at both higher l e v e l s of S had increased N uptake at higher temperatures. Rape plants grown at 0 ppm S l e v e l were not affected by temperature excepting at 35/27°C where the t o t a l N uptake was considerably higher than at other temperatures (Tables 8 and 9). Nitrate Nitrogen Concentration Concentrations of NOg-N i n the shoot tissue of a l l three species at various S l e v e l s within temperature regimes were not s i g n i f i c a n t l y d i f f e r e n t , excepting at the lowest S l e v e l i n barley grown at 18/10°C. This was s i g n i f i c a n t l y lower than the concentration at the higher S l e v e l s . Barley showed greater NO^-N concentration at lower S l e v e l s at 29/21 than at 24/16°C and greater NO^-N concentration at higher S l e v e l s at 24/16 than at 18/10 or 29/21°C. Peas had the highest N03~N concentration for a l l three S l e v e l s at 29/21°C. Rape had the highest concentration at 24/16 for 64 ppm and at 29/21°C for 0 and 8 ppm le v e l s (Table 8). 93 Total Nitrate Nitrogen Uptake Total NO^-N uptake as affected by temperature was si m i l a r to the N concentration i n barley, peas and rape shoot t i s s u e . Generally the t o t a l uptake was greater i n plants grown with adequate S than under d e f i c i e n t conditions. N:S Ratio As shown i n table 8, s i g n i f i c a n t l y d i f f e r e n t N:S ra t i o s were obtained f o r S l e v e l s within temperature regimes i n barley. Usually the highest r a t i o s were recorded f o r the lowest S l e v e l and r a t i o s gradually tended to decrease with increase i n temperature. This trend which was observed f o r a l l l e v e l s i n barley was most pronounced at the 0 ppm l e v e l . N:S r a t i o s f o r peas at 0 ppm were greatest at 29/21°C. At 8 and 64 ppm these r a t i o s tended to be the same at the three temperature regimes. Lowest N:S r a t i o f o r a l l three l e v e l s was at 24/16°C. Ratios for rape at 0 ppm were also greatest at 29/21°C. At 8 ppm they were maintained at the same l e v e l at a l l three temperature regimes, while at 64 ppm they tended to decrease s l i g h t l y with increase i n temperature. E f f e c t of Temperature and Sulphur N u t r i t i o n on the Y i e l d and  Mineral Concentration i n F r u i t of Barley, Peas and Rape Dry weights of f r u i t s of barley and peas were s i g n i f i c a n t l y greater at the higher S l e v e l s than at the lowest S l e v e l at a l l three temperature regimes as given i n table 10. If the growing temperature was increased above 18/10°C, the Table 10. E f f e c t of temperature and sulphur n u t r i t i o n on the y i e l d , sulphate sulphur and nitrogen concentration i n the f r u i t s of barley, peas and rape. Experiment 5 Temperature Sulphur day/night l e v e l Species ^2 (ppm) Barley Peas Rape 18/10 24/16 29/21 18/10 24/16 29/21 18/10 24/16 29/21 0 8 64 0 8 64 0 8 64 0 8 64 0 8 64 0 8 64 8 64 8 64 8 64 F r u i t dry weight g  ft 0.05b 5.63a 4.92a 0.003b 2.46a 2.49a 0.01b 0.77a 0 .96a 0.2 8b 5 .53a 5.51a 0.4 5b 2.91a 2.71a 0.27b 1.30a 1.70a 0 . 38a 0.17b 0.47a 0 .16b 0 .07a 0 .06a Percent of dry matter SO^ 0.016b 0.014b 0.092a 0.001b 0.034b 0.097a 0.001b 0.061b 0.092a 0.036a 0.048a 0.054a 0.003b 0.039a 0.048a 0.004b 0 . 036a 0.043a 0.09b 0 .39a 0.05b 0 .38a 0.05b 0.46a N N 0.95b 1.94a 2 .04a 0. 81b 1.75a 1.90a 0 .67b 1, 2, 3, 2, 2, 4, 2, 2 , 4, 3, 3, 2. 2 , 2, 2, 2, 1. 96a 11a 78a 53b 66b 44a 90b 59b 67a 65ab 17b Ola 76a 05a 15a 32a 7 3a Total uptake per plant (mg) §°4 0 .009c 0.790b 4.526a 0.001c 0.083b 2 .420a 0 .001b 0.469ab 0 .699a 0.100b 2.640a 2.972a 0.013b 0.553b 300a 011a 0 . 338a 0.731a 1 0 0.365a 0.663a 0.235b 0 .608a 0.035b 0.276a 0.47b 109.46a 100.51a 0.02b 43.05a 46.95a 0.067b 15.09ab 16.26a 10.58b 139.94a 146.56a 19.64b 84.41a 69.82a 13.90b 47.97a 54.36a 7 .65a 4.69a 10.46a 3.44b 1.62a 1.05a Percent of t o t a l shoot weight 7.6b 50 . 5a 47 .3a 0.4b 36.6a 39.2a 1.2b 19.2a 20.2a 20.4b 41.4a 45.3a 34.6b 51.5a 48 .9a 25 .0b 44.8a 49 .4a 6.2a 2.8b 7 .8a 2.9b 1.1a 0.8a * Each figure i s the mean of 6 r e p l i c a t e s . Figures followed by the same l e t t e r within a species, temperature regime and y i e l d or element are not s i g n i f i c a n t l y d i f f e r e n t at P = 0.0 5 according to Duncan's new multiple range t e s t . 95 y i e l d s of barley heads and pea pods were decreased at a l l three l e v e l s . Rape had greater weight at 8 ppm than at 64 ppm. Highest y i e l d was obtained at 24/16°C. Sulphate Sulphur Concentration Concentration of SO^-S i n barley heads at 0 and 8 ppm S l e v e l s at the three temperature regimes was s i g n i f i c a n t l y lower than at the highest S l e v e l . A temperature x S l e v e l i n t e r a c t i o n was evident i n the data f o r barley and peas. The lowest l e v e l at 18/10°C f o r barley and peas was 16 and 12 times greater than at 24/16°C. On the other hand, concentration at 8 ppm i n barley, tended to increase by a f a c t o r of about 2 f o r each increased temperature regime, whereas i n peas there was a s l i g h t decrease from 18/10 to 24/16°C. Concentration at the highest l e v e l was not affected by temperature i n barley and peas. Data f o r rape was available only for 8 and 64 ppm, since no pods were developed at 8 ppm S l e v e l . S i g n i f i c a n t differences i n the SO^-S concentration between these two l e v e l s were observed. There was no e f f e c t of temperature on S concentration at 8 ppm, but at 64 ppm concentration was s l i g h t l y more at 29/21°C than at the two lower temperature regimes. Average SO^-S concentration i n the seeds of barley, peas and rape were 0.024, 0.044 and 0.389 re s p e c t i v e l y . Total Uptake of Sulphate Sulphur Total uptake of SO^-S i n the f r u i t s of the three species were affected by both l e v e l s and temperature regimes. S i g n i f i c a n t l y lower uptake was observed at the lower S l e v e l 96 than at the two higher S l e v e l s i n barley and peas, at a l l temperature regimes excepting i n peas at 29/21°C. Total SO^-S uptake i n rape pods was greater at 64 ppm than at 8 ppm S. Increase i n temperature caused a reduction i n the t o t a l uptake at the three l e v e l s i n a l l three species (Table 10). Nitrogen Concentration N concentration i n barley heads increased with increase i n the supply of S and s i g n i f i c a n t differences between the lowest and the higher l e v e l s were present as shown i n table 10. Temperature had l i t t l e or no e f f e c t on the N concentration. With increased S a p p l i c a t i o n , N concentration i n the pods of pea plants tended to decrease. These differences were found to be s i g n i f i c a n t . Increase i n N concentration due to increase i n temperature was observed at a l l l e v e l s , i n pea plants. This increase was not as great as the difference due to S l e v e l s . Rape pods were lea s t affected by temperature at both l e v e l s . Total Uptake of Nitrogen S i g n i f i c a n t differences between l e v e l s were observed i n the t o t a l uptake of N i n barley heads. Greatest increase occurred at the highest S l e v e l . There was a decrease i n the t o t a l uptake with increase i n temperature. Peas also had s i m i l a r s i g n i f i c a n t differences between S l e v e l s . Total uptake i n pea pods decreased with increase i n temperature at the three l e v e l s . 97 S i g n i f i c a n t l y higher uptake of N by rape plants at 64 ppm than at 8 ppm S l e v e l was observed at 24/16°C. F r u i t Weight as Percent of Total Dry Weight At the lowest l e v e l of S f r u i t s of barley and peas contributed a s i g n i f i c a n t l y lower percentage of the t o t a l weight compared to the two higher l e v e l s as given i n table 10. Percentages at both 8 and 64 ppm were about the same. Contribution from barley heads was less at higher temperature for a l l l e v e l s . Pea pods were l e a s t affected by temperature and the percentages tended to be about the same at the three temperature regimes. Rape pods contributed a small percentage of the t o t a l weight. Highest percentages were recorded f o r both l e v e l s at 24/16°C. E f f e c t of the Removal of Cotyledons or Endosperm on Sulphur Responses of Barley, Peas and Rape Growth Barley showed l i t t l e or no change by the removal of endosperm with respect to shoot and root lengths (Table 11). S i g n i f i c a n t differences between S l e v e l s within both treatments were evident. Shoot lengths of peas and rape were reduced by the removal of cotyledons, but there were no s i g n i f i c a n t differences between S l e v e l s within treatments. Generally the weights of shoots and roots were reduced due to the removal of cotyledons and the e f f e c t of S n u t r i t i o n was n e g l i g i b l e or absent (Table 11). Table 11. E f f e c t of excision of cotyledons or endosperm and sulphur n u t r i t i o n on the growth and y i e l d factors of barley, peas and rape at 4 weeks (vegetative stage)"*". Experiment 6 Treatment Species (Cotyledons) Sulphur l e v e l (ppm) Shoot length cm Root length cm Shoot fresh weight Root fresh weight g Shoot dry weight Root dry weight g Barley Intact 0 50, ,5b 22. . 3ab 11. ,6b 4, .lb 1. ,45b 0. ,47b 8 67, . 8a 26, . 5a 27. ,2a 11, , 0a 3, .Ola 1. ,14a 64 66 , . 3a 26. . 5a 25. .2a 10, . 3a 2, .89a 0. ,97a Excised 0 53, ,5b 24. . 3a 13. . 6a 4, . 5a 1, .18b 0. , 30b 8 66 , .4a 27, . 3a 19. ,9a 5, .7a 1, . 80ab 0. , 37b 64 62, ,0a 25, .4a 19. ,9a 7, .la 2, .03a 0. ,54b Intact 0 37, . 3a 25. .0a 17. ,9b 17 , .7a 2. .05b 1. .11a 8 41. ,7a 23. ,2a 26, . 3a 19. .4a 3, .16a 1. , 20a 64 42, ,2a 24. , 3a 27. ,2a 17, , 6a 3, .27a 1. ,08a Excised 0 21. .7a 24. ,1a 8, ,0a 7, . 6a 0. ,84a 0 , .42a 8 26, ,0a 21. , 6a 10. .la 5, . 8a 1. .03a 0. . 33a 64 26, ,4a 21. .2a 11, .4a 4, . 8a 0. .79a .0. .23a Intact 0 20.7ab 22.5a 15.7ab 2.7a 1.44a 0.18a 8 20.7b 13.25bc 11.5b 2.0a 0.69bc 0.16a 64 24.5a 14.2bc 20.5a 1.6a 1.35ab 0 .16a Excised 0 14.4bc 15.4b 0. 8c 0.1a 0 .06c 0.01a " 8 12.6c 9.7c 2.6c 0.2a 0.17c 0.02a 64 17.5abc 10.5c 3. 6c 0.2a 0. 35c 0.02a + Plants were grown at 24/16°C * Each figure i s the mean of 3 r e p l i c a t e s . Figures followed by the same l e t t e r within a species and growth measurement are not s i g n i f i c a n t l y d i f f e r e n t at. P = 0.05 according to Duncan's new multiple range test. 99 Nodes S i g n i f i c a n t differences between S l e v e l s were eithe r few or completely absent i n the three species for both treatments (in t a c t and excised). An o v e r a l l decrease i n the number of nodes was observed i n the treatments i n which cotyledons or endosperm were removed. This e f f e c t was greater i n peas and rape than i n barley according to data given i n table 12. E f f e c t of Temperature and Sulphur N u t r i t i o n on Net CO,, Exchange  Rates of Barley, Peas and Rape at 2 0 and 30 Days As shown i n table 13 highly s i g n i f i c a n t differences were usually evident among measuring temperatures and S l e v e l s i n a l l three species for the three methods of expressing C0 2 exchange rates. Highly s i g n i f i c a n t differences among growing temperatures were observed for barley at 20 days and for peas at 30 days. Barley at 30 days and peas and rape at 20 days also had s i g n i f i c a n t differences for most of the measurements. Interactions between growing temperatures and measuring temperatures, growing temperatures and S l e v e l s and measuring temperatures and S l e v e l s were also found i n a l l three species. In peas and rape, these interactions occurred more often i n 30 day old plants, whereas i n barley they occurred mostly i n 20 day old plants. The most consistent measurement was found to be on the basis of l e a f area rather than dry weight or fresh weight. 100 Table 12. E f f e c t of excision of cotyledons or endosperm and sulphur n u t r i t i o n on the growth of nodes of barley, peas and rape at 4 weeks (vegetative stage)"4". Experiment 6 Treatment ^ e ^ e l 1 " Growth stage (days from planting) Barley Peas Rape (Cotyledons) (ppm) 14 17 20 23 26 29 Intact 0 ft 2 . 5a 3, .2a 3 , 8b 4, . 3b 4, ,9b 5 , .0b 8 2. ,7a 3 , . 5a 4, , 3a 5 , 2a 5, . 6ab 6 , 4a 64 2. . 9a 3, . 5a 4. . 5a 5. , 3a 6 , . 0a 6 , .4a Excised 0 2. , 5a 3, .4a 3. ,9b 4. , 5b 5. ,1a 5 , 5b 8 2, ,7a 3 , . 6a 4. , 5a 4, . 9ab 5, . 5a 5, . 9ab 64 2. , 5a 3, . 8a 4, . 6a 5, . 3a 5, ,7a 6, ,8a Intact 0 3, . 6a 4, . 5a 5. . 8a 6. . 9a 8. . 5a 9 , .la 8 3, ,7a 4, . 9a 6. . 0a 7, .la 8 . 7a 9 , .4a 64 3, ,7a 4, ,7a 6, ,0a 7, .2a 8, , 6a 9 , .3a Excised 0 2, . 5a 3 , . 5a 4, . 6a 5, . 6a 6, . 3a 7 , .la - 8 2, . 5a 3, .4a 4. , 3a 5, , 3a 6, , 3a 7, . 0a 64 2, , 3a 3, . l a 4. ,2a 5 , .2a 6, , 3a 6 , .9a Intact 0 2, ,1a 2, .7a 3 , . 5a 4. ,2a 5 , 2a 5, . 5a 8 2, . 0a 2, ,7a 3, . 8a 4, . 9a 5, , 3a 5, . 8a 64 2. .2a 2, .7a 3. , 8a 4. . 6a 5, . 5a 5, .7a Excised 0 1, ,9a 2, ,2a 3, ,0a 3, .7a 4, . 6a 4, . 8a 8 1, ,9a 2 , .l a 3 , 0a 3 , . 9a 4, . 5a 5, .0a 64 1. .7a 2 , . 0a 3, . 0a 3 , .7a 4, . 6a 4, . 8a + Plants were grown at 24/16°C * Each figure i s the mean of 3 r e p l i c a t e s . Figures followed by the same l e t t e r , within a species, and growth stage are not s i g n i f i c a n t l y d i f f e r e n t at P = 0.05 according to Duncan's new multiple range t e s t . Table 13. F values and the significances of main effects and interactions of net C0 o exchange rate for barley, peas and rape. Experiment 7 20 Day 3 0 Day mg C0 2/ mg c o 2 / mg C0 2/ mg C0 2/ mg C0 2/ mg C0 2/ hr/dm^ hr/ gdw hr/gfw hr/dm^ hr/gdw hr/gfw B a r 1 e y Growing temperature (G) 15.50** 29. 75** 18.80** 1.95 ns 2.72 ns 3 .80* Measuring temperature (M) 34.81** 29. 04** 30.72** 23 . 83** 8.81** 3 0.83** Sulphur l e v e l (S) 92.05** 139. 59** 43.10** 51.72** 24.50** 2 7.98** G x M 3 .45** 2. 7 3 * * 3.64** 1.0 7 ns 1.50 ns 1.54 ns G x S 1.44 ns 3 . 31* 1.69 ns 3.61* 0.64 ns 0.22 ns M x S 2.0 8 ns 3 . 14* 1.25 ns 2.89* 3.26** 2.18 ns P e a s -Growing temperature (G) 4.36* 3. 23* 1.62 ns 7.9 6** 9.19** 16.82** Measuring temperature (M) 70.78** 67. 06** 44.14** 95.06** 54.28** 54.96** Sulphur l e v e l (S) 9.02** 5. 45* 1.96** 23.55** 14.5 8** 21.47** G x M 1.8 5 ns 1. 58 ns 1.25** 9.0 0** 7.67** 9.64** G x S 0.07 ns 0. 26 ns 0.46 ns 0.55 ns 0.26 ns 0.40 ns M x S 1.19 ns 1. 7 6 ns 0.71 ns 4.8 2** 1.36 ns 1.28 ns R a P e Growing temperature (G) 0.32 ns 4. 16* 4.35* 24.9 3** 3.51* 11.24** Measuring temperature (M) 22.68** 19. 06** 18.23** 30.69** 30.81** 19 .72** Sulphur l e v e l (S) 6. 33* 11. 50** 6.45* 66.38** 51.47** 40.43** G x M 2 .42** 1. 65 ns 1.46 ns 2.22* 2.46** 1.99 ns G x S 1.12 ns 0. 6 3 ns 0.2 8 ns 7.0 3** 3.95* 2.57 ns M x S 1.88 ns 0. 22 ns 0.41 ns 3.58** 6.74** 2.64* 102 Plants grown at a l l temperature regimes with 64 ppm S had s i g n i f i c a n t l y greater C0 2 exchange rates than with 0 ppm 2 S i n a l l three species when expressed as mg CC^/hr/dm , mg C02/hr/gdw or mg C02/hr/gfw (Table 14, 15 and 16). The response to S n u t r i t i o n at d i f f e r e n t measuring temperatures had the same trend f o r a l l three methods of expression f o r a p a r t i c u l a r species, although there were differences i n the magnitude of the response. Where barley plants are grown at 18/10°C, and measured at various temperatures, at 20 days, highest net C0 2 exchange rates were observed between 7 and 18°C. This same pattern was followed by 30 day old plants. Barley plants grown at 24/16°C temperature regime had maximum rates at measuring temperatures between 13 and 24°C f o r both 20 and 30 day old plants. I f they were grown at 29/21°C t h e i r maximum exchange rates were between 18 and 29°C f o r both 20 and 30 day old plants. I f they are grown at 35/27°C t h e i r maximum exchange rates were at measuring temperatures between 24 and 35°C. In other words maximum C0 2 exchange rates were recorded at or below the growing temperatures. Peas followed the same pattern as barley with regard to the response to growing and measuring temperatures. Rape plants had a s i m i l a r pattern of response to that of barley and peas. However, i n rape the peak of maximum C0 2 exchange rates was spread over a wider range of measuring temperatures, i n d i c a t i n g a wider range of a d a p t a b i l i t y . Table 14. E f f e c t of temperature and sulphur n u t r i t i o n on net CO exchange rates of barley at two growth stages. Experiment 7 Growing temperature Measuring Sulphur day/night temperature l e v e l °C ^C (ppm) 18/10 18 0 64 13 0 64 7 0 64 18 0 64 24 0 64 29 0 64 24/16 24 0 64 18 0 64 13 0 64 24 0 64 29 0 64 35 0 64 2 0 Day mg C0 2/ mg c o 2 / mg C0 2/ hr/dm2 hr/ gdw hr/gfw 16.4cd 27. 2d 3 .2de 24.8a 53. l a 4.7a 16.5cd 26. 6d 3.2de 23 .7a 50. 3ab 4.5ab 17.4cd 27. 9d 3.4cde 19.2bc 43. 7bc 3.9abcd 18.1c 29. 6d 3.2de 23 ,8a 52. 2a 4.6a 16.3cd 26. 8d 3.1de 21.9ab 49. lab 4.3abc 14.0a 23. 4d 2.7e 18.8bc 40. 6c 3.6abcde 18.9de 45. 9efg 3.8cde 29 .24a 66. 3b 5.7a 19 .9cde 48. 4def 4.1bcd 32 .27a 74. 9a 6 . 3a 21.1bcde 51. 7cde 4.2b 2 8.4a 65. 2b 5 . 6a 21.3bcd 39. 6gh 4.0bcd 30.6a 71. 4ab 5.9a 17.9ef 44. 2fgh 3 .4de 23 .lbc 54. led 4.7b 15.If 38. l h 2 .9e 24.1b 56. 2 4.6b 3 0 Day mg C0 2/ mg c o 2 / mg c o 2 hr/dm2 hr/ gdw hr/ gfw 10.7c 17. lb 2. lb 28.1a 34. l a 3. 7a 10.4c 16. 7b 2. lb 28.0a 34. 5a 3. 9a 9.3c 15. 9c 1. 9b 26.0a 33. 8a 3 . 8a 10.7c 17. 7b 2 . 2b 26.0a 33. 9a 3. 9a 10.1c 16. 9b 2 . lb 24.9ab 32. 7a 3. 9a 8 .6c 15. 4b 1. 9b 21.4b 28. 4a 3. 3a 10.8d 9. 9d 1. 5e 29.0a 26. 0a 4. 7a 11. 7d 10. 6cd 1. 6e 2 3.6b 25. Oab 3 . 5bc 10.9d 10. l d 1. 5e 24.9b 25. 8a 3 . 7b 10. 5d 9. 7d 1. 5e 23.2b 25. l a 3 . 6bc 9.7d 8. 8d 1. 4e 18.3c 19. 8ab 2. 9cd 7.5d 6. 9d 1. Oe 15.6c 17. 4bc 2 . 5d Table 14. (continued) Growing temperature Measuring Sulphur day/night temperature l e v e l °C °C (ppm) 29/21 29 0 64 24 0 64 18 0 64 29 0 64 35 0 64 41 0 64 35/27 35 0 64 29 0 64 24 0 64 35 0 64 41 0 64 46 0 64 2 0 Day mg C0 2/ mg C0 2/ mg C0 2/ hr/dm2 hr/gdw hr/gfw it 15.5cd 30.6e 3.9cd 25.6ab 56.8ab 6.8ab 14. Id 27.9e 3 ,6d 27 .7a 59 .6a 7.3a 13.Id 25.7e 3.3d 26.2ab 5 8.1a 6.9ab 15.Id 30.2e 3.9cd 2 5.3ab 55.9ab 6 ,7ab 12. 8d 25. l e 3 .3d 22.9b 50.5b 6.1b 11.9d 23. 3e 3.0d 17.7c 38 .9c 4.6c 17.9g 29.7f 4.8f 29 .5bc 46.7abc 7.4bc 2 8.8bcd 33 .Oef 5.1e 31.7ab 50.4ab 7.9ab 26.3cde 37.4de 5.5ef 32.3ab 51.0ab 8 .lab 23,3ef 3 8.4de 5.2ef 33.7a 53.2a 8.4a 20.8fg 34.8ef 4.5f 27.6cd 43.5bcd 6 .9cd 13.69h 22.6g 3.6g 25.6de 40 .4cde 6 .4d 3 0 Day mg c c y mg c c y mg C0 2/ hr/dm2 hr/ gdw hr/gfw 10 .led 10. 4c 1.5cd 18 . 5ab 26. 2a 3.3a 11 .2c 11. 5c 1.6cd 20 ,9a 29. 7a 3 .7a 10 .led 10. 4c 1.5cd 20 .9ab 29. 8a 3 .7a 10 .4cd 10. 7c 1.5cd 18 . 5ab 26. 3a 3 . 3a 8 . 6cd 8. 9c 1.3d 16 .3b 23. lab 2.9ab 6 .4d 6. 6c 0.9d 12 . 5c 18. 8b 2 .2bc 14 .7abcd 13. 8ef 3.8cde 17 .2ab 23. 8abcd 4.4bc 14 . 3bcd 17. 9cde 3 . l e f 17 .4ab 27. 5ab 4.9ab 13 . 8bcd 16. 9de 3 .12ef 18 . 6a 29. 8a 5 . 3a 11 . 9de 14. 4ef 2.7fg 15 . 6abc 24. 7abc 4.4bc 12 . 6de 17. 9cde 3 .2ef 14 .8abcd 20. 6bcde 3 . 7cde 7 .2e 8. 7f 2.0g 11 . 8cd 18. 8cde 3.4de * Each figure i s the mean of 3 r e p l i c a t e s . Figures followed by the same l e t t e r within a growing temperature regime and a p a r t i c u l a r measurement are not s i g n i f i c a n t l y d i f f e r e n t at P = 0.05 according to Duncan's new multiple range t e s t . Table 15. E f f e c t of temperature and sulphur n u t r i t i o n on net CO exchange rates of peas at two growth stages. Growing temperature Measuring Sulphur day/night temperature l e v e l °C °C (ppm) 18/10 18 ^ 0 -64 13 0 64 7 0 64 18 0 64 24 0 64 29 0 64 24/16 24 0 64 18 0 64 13 0 64 24 0 64 29 0 64 35 0 64 2 0 Day mg c o 2 / mg C0 2/ mg C0 2 hr/dm2 hr/gdw hr/gfw 22 .7ab 51.8bc 5.9abc 23 . 9ab 58.90ab 6.40a 23 . 5ab 55.4abc 6.3a 25 .0a 60.0a 6. 5a 21 . 3bc 52.4abc 5.7abc 24 ,9a 56.8abc 6 .4a 22 . 8ab 65 . 3abc 6. lab 25 .7a 59. 9a 6. 6a 18 . 8cd 48.9c 5.Obcd 23 • lab 50.0c 5.9abc 16 .Id 41.3d 4.3d 18 .9cd 41.7d 4.9cd 16 .5de - 42.4d 4.7de 19 . 8bc 50.6bc 6.1bc 17 .9cd 45.8cd 5 .led 24 . 6a 62 .6a 7.6a 18 . 3cd 46.5cd 5.2cd 23 . 8a 60.2a 7.3a 17 . Ocde 43 .4cd 4.9d 22 . Oab 55.9ab 6. 8ab 14 .6ef 37.6de 4.2e 17 . Ocde 43.4cd 5.3cd 12 .8f 31.9e 3.6e 14 .7ef 37.4e 4.5de 3 0 Day  mg C0 2/ mg C0 2/ mg C0 2/ 2 hr/dm hr/gdw hr/gfw 7 .6bc 13 . 5cd 1. 7dc 10 . 36a 18 •12abc 2. 7abc 8 .2b 14 .7bcd 1. 8de 10 .9a 19 .8 3 . 0a 6 . 8bc 14 . Ocd 2. Ocde 12 .2a 21 .2a 3. 2a 7 . 2bc 12 .7d 1. 6de 12 . 0a 21 .7a 3 . 3a 6 . 8bc 12 .3d 1. 5e 10 .4a 18 .7abc 2. 9ab 5 .8c 10 .4d 1. 5e 8 .3b 15 .Obcd 2. 3bcd 7 .8c 11 .9b 1. 6b 12 . 6ab 19 .7a 2. 6a 7 • 2c 11 .0b 1. 5b 14 . 3a 22 . 5a 2. 9a 6 .5cd 10 .0b 1. 3b 14 .2a 22 • l a 2. 9a 6 .4cd 10 .0b 1. 3b 12 .7ab 20 • l a 2. 6a 6 .lde 10 . 8b 1. 4b 10 .9b 17 .4a 2. 3a 4 ,9d 7 ,6b 1. 0b 7 . l c 11 .2b 1. 5b Table 15. (continued) Growing temperature Measuring Sulphur day/night temperature l e v e l °C (ppm) 29/21 29 0 64 24 0 64 18 0 64 29 0 64 35 0 64 41 0 64 35/27 35 0 64 29 0 64 24 0 64 35 0 64 41 0 64 46 0 64 2 0 Day  mg C0 2/ mg C0 2/ mg C0 2/ 2 hr/dm hr/gdw hr/gfw 15 .7de 34 . l e f 4 . 5cd 18 . 3cd 42 .4bcd 5 . 9ab 17 .6cd 39 . 9de 5 . 2bc 22 .9a 50 .4ab 6 . 6a 18 .6cd 41 . 5cde 5 .4bc 22 .4ab 52 ,9a 6 . 9a 16 .9cd 40 . 5cde 5 • 2bc 19 .9bc 47 . 8abc 5 .9ab 13 .3ef 29 .5f 4 . 5cd 16 .9cd 35 .ldef 5 .2bc 9 •0g 19 •9g 2 . 6e 11 • 5fg 27 .7e 3 .7de 15 .Odef 39 • 4efg 5 . 3efg 19 .lbc 47 .4abcd 6 .2cdef 17 . 7cde 46 .lbcde 6 . 3cde 21 .4ab 53 . 5ab 7 . Oabc 20 . labc 52 .7abc 6 • 7bcd 22 . 8a 55 .0a 7 .7ab 17 . 8cd 43 .3def 5 .8defg 22 . 3a 52 . 9abc 8 • l a 15 .7def 39 .9def 5 •2fg 17 . 8bcd 45 .3cdef 5 . 9cdefg 13 .14f 31 •9g 4 .lh 14 .7ef 37 • 6fg 4 • 9gh 3 0 Day  mg C0 2/ mg C0 2/ mg C0 2/ 2 hr/dm hr/gdw hr/gfw 9 .led 16 .9ef 2 . 3cd 12 .9b 24 . 3cd 3 .4b 9 . 9cd 18 . 8e 2 .6 15 .7a 29 . 6ab 4 .lab 10 .led 19 .le 2 . 6c 16 . 6a 31 . 3a 4 .4a 8 . 6de 16 .2ef 2 .2cd 13 .3b 25 .lbc 3 .5b 6 .9e 12 •7fg 1 . 8de 10 ,7c 19 . 9de 2 .8bc 4 .8f 9 •lg 1 . 3e 8 .9cd 16 .9ef 2 .4cd 12 .9ef 25 .2d 3 . 9de 14 .4de 30 .7c 4 . 5cd 16 .2bcd 25 .5d 3 .9de 16 .7b 35 . 2abc 5 . 3ab 17 .3b 36 . 6ab 5 .7a 20 . 5a 39 . l a 5 . 8a 14 . 7cde 31 .Id 4 . 6bc 16 . 5bc 31 . 9bc 4 . 9bc 11 .56f 22 .6d 3 .4e 12 .2f 25 .6d 3 . 9de 4 •3g 7 .9f 1 •lg 11 ,7f 16 . 9e 2 .2f * Each figure i s the mean of 3 r e p l i c a t e s . Figures followed by the same l e t t e r within a growing temperature regime and a p a r t i c u l a r measurement are not s i g n i f i c a n t l y d i f f e r e n t at P = 0.05 according to Duncan's new multiple range t e s t . Table 16. E f f e c t of temperature and sulphur n u t r i t i o n on net CO exchange rates of rape plants at two growth stages. Experiment 7 Growing temperature Measuring Sulphur mg C O 2 / day/night temperature l e v e l 2 °C °C (ppm) hr/dm 2 0 Day m. g c o 2 / hr/gdw mg C 0 2 / hr/gfw 3 0 m hr/dm Day g C 0 2 / mg C 0 2 / 2 mg C 0 2 / hr/gdw hr/gfw 18/10 24/16 18 - 0 20.4cd 36.1def 3 .6cd 25.5bc 31.3cd 3.2c 64 29.3ab 52.6ab 4.9ab 30.2a 41.7a 4.2a 13 0 21.8c 37.2de 3.71cd 26.7bc 32.7cd 3 .3bc 64 32 .3a 57.6a 5. 5a 30.3a 41.7a 4.2a 7 0 25.8ab 42.6cd 4.3bc 26 .2bc 32.3cd 3. 3bc 64 31.2a 56.2ab 5.3a 30.4a 41.9a 4.2a 18 0 19.led 37.5de 3.4cd 26.7bc 32.8c 3 . 3bc 64 31.0a 55.9ab 4.9ab 29.9a 41.4ab 4.1a 24 0 17.2d 31.5ef 3. Ode 26.4bc 32.6cd 3 . 3bc 64 26.2ab 53.8ab 5.2a 30. 0a 41.7a 4.2a 29 0 13.Se 28. 3f 2.5e 23.8c 29.4d 3 .0c 64 22.3ab 47.5bc 5.0ab 27 .3b 37.2b 3 .7ab 24 0 23. Ocde 42.3de 4. Ode 13. 2d 10.3d 1.4c 64 25.6abc 55.4ab 4. 9abc 27.2ab 44.4a 3 ,4a 18 0 25.Oabcd 44. Ocde 4.6abcd 13. l d 10.3d 1.4c 64 28.5a 55.4ab 5.3a 27.9a 45.7a 3 . 5a 13 0 23 .lede 42.3de 4. Ode 11.7d 9.0d 1.3c 64 25.9abc 56.lab 5.0ab 2 5.8ab 44.1a 3 .2a 24 0 23.5bcde 42.Ode 4.Ocde 13.4d 10.4d 1.5c 64 27.3ab 59.3a 5.3a 26.3ab 42.9ab 3 .2a 29 0 21.2def 41.11de 3 . 9de 11.9d 9.2d 1.3c 64 23 . 9bcde 49.5bcd 4.4bcd 24.2b 39.5b 3 .0a 35 0 18.9f 36.2e 3. 3e 8 .9e 6.9d 0. 8c 64 20.9ef 45.7cde 4.lede 19 .5c 31.9c 2 ,4b o ^3 Table 16. (continued) Growing temperature Measuring Sulphur day/night temperature l e v e l °C °C (ppm) 29/21 29 0 64 24 0 64 18 0 64 29 0 64 35 0 64 41 0 64 2 0 Day mg c o 2 / mg c o 2 / mg C0 2/ hr/dm2 hr/gdw hr/gfw 24 .7abc 39 . 6c, 4.6abc 26 . 3ab 71 .9a 5.2a 23 .8abcd 38 . 3c 4.5abc 26 . 8a 73 .4a 5.3a 22 .03cd 34 .2cd 3.9cde 22 .8abcd 64 . 8ab 4.7ab 22 . 6bcd 37 .4c 4.4abcd 24 . 9abc 65 . 8ab 4.7ab 20 . 3de 32 .led 3 . 8de 23 .5abcd 64 . 9ab 4.7ab 17 ,7e 27 .3d 3.2e 20 .4de 58 .6b 4.2bcd 3 0 Day  mg C0 2/ mg C0 2/ mg C0 2/ 2 hr/dm hr/gdw hr/gfw 11.9def 9.4d 1.53d 24.6a 46.3a 3.6ab 13.0cd 7.6de 1.7d 25.0a 46.9a 3.7ab 10.3ef 6.2de 1.8d 25.3a 47.2a 3.8a 10.7ef 6.4de 1.4d 23.7ab 44.6ab 3.5ab 5.6fg S.le 0.8e 21.6b 40.6b 3.2b 4.7g 3.8e O.le 7.8b 33.5e 2.6c 35/27 35 0 20.3ef 57.3cd 5.7de 10. 9f 21.7e 2 . 5e 64 20.9ef 76.8a 6 . 3bcd 18.9ab 42.9b 3 . 9ab 29 0 22.4cde 67.5b 6 . Ocde 14.8cd 29.8d 3 .2a 64 28.3a 84.3a 7 .2ab 19.8a 45.0ab 4 .3a 24 0 21.9de 66.2bc 6.Ocde 14.9cd 30. Od 3 .Id 64 25.3abcd 82.9a 7.5a 21.2a 48.5a 4 .4a 35 0 2 2.7bcde 67.4b 5. 9cde 16.Ocd 31.4d 3 .7bc 64 27.lab 82.6a 6.8abc 19.8a 44.9ab 4 .4a 41 0 22.1de 64.8bc 5 . 6de 13.6de 24.4e 3 .ldc 64 26.labc 80.7a 6.6abc 16.8bc 41.1b 3 . 5bcd 46 0 17.6f 52.ld 4.7f 11.9ef 21.6e 2 .4c 64 20.1ef 62.4bc 5.2ef 14.9cd 36.2c 3 .5bcd * Each figure i s the mean of 4 r e p l i c a t e s . Figures followed by the same l e t t e r within a growing temperature regime and a p a r t i c u l a r measurement are not s i g n i f i c a n t l y d i f f e r e n t at P = 0.05 according to Duncan's new multiple range t e s t . 109 Table 17 gives the r a t i o s of CC^ exchange rates f o r S s u f f i c i e n t and d e f i c i e n t plants at 20 and 30 days, when meas-ured at the growing temperature and 5.5°C higher than the growing temperature. Generally, there was an increase i n the r a t i o s at 30 days. In other words, S n u t r i t i o n became more important at 3 0 days than at 2 0 days for a l l the three species. However, t h i s trend was reversed f o r barley and peas at 35/27 and for rape at 18/10°C. Generally when measurements were taken at growing temperatures which are cl o s e r to optimum growing temperatures, the rates tended to be higher than those taken at higher temperatures. Unless a high growing temperature .is not too detrimental f o r growth, the r a t i o s of CC>2 exchange rates f o r S s u f f i c i e n t and d e f i c i e n t plants obtained when measured at that temperature tended to be s l i g h t l y higher than those obtained for plants measured at the same temperature but grown at a lower temperature regime. For example, the figures 1.34 f o r 20 day old 18/10 barley plants and 1.54 for 24/16 plants measured at 24°C, or 1.29 for 24/16 plants and 1.65 for 29/21 plants measured at 29°C may be compared (Table 17). E f f e c t of Temperature and Sulphur N u t r i t i o n on CO,, Compensation  Points i n Barley, Peas and Rape Highly s i g n i f i c a n t differences i n compensation points between temperature and between l e v e l s within temperature regimes were obtained (Table 18). Also s i g n i f i c a n t temperature by S l e v e l i nteractions were observed f o r both CC^ compensation point and percent t o t a l S. Table 19 gives the s i g n i f i c a n t 110 Table 17. Ratios of mg CC^/hr/dm exchanged i n plants grown with 64 ppm sulphur and 0 ppm sulphur. Measurements compared at the growing temperatures and at 5.5°C higher. Experiment 7 Temperature regime Measuring day/night temperature Barley Days from planting Peas Days from planting Rape Days from planting °c °C 20 30 20 30 20 30 18/10 18 1. ,51 2 . ,64 1. ,05 1. ,36 1. ,43 1. .18 24 1. ,34 2. .46 1. .23 1. ,52 1, .50 1. ,13 24/16 24 1. ,54 2. .68 1. .19 1. ,60 1. .11 2. .06 29 1. ,29 1, .88 1, .17 1, .53 1. .12 2, .03 29/21 29 1. ,65 1, .82 1, .16 1, .41 1, .06 5, .29 35 1. .79 1, .88 i , .26 1, .55 1, .15 3 , .63 35/27 35 1. ,64 1, .23 1, .27 1. .12 1, .03 1, .74 41 1. .32 1, .16 1, .13 1. .05 1, .18 1, .23 Table 18. F values and significances of main effects of temperature and sulphur l e v e l and t h e i r i n t e r a c t i o n . Experiment 8 Barley Peas Rape C O 2 C 0 2 c o 2 Compensation Percent Compensation Percent Compensation Percent Treatment point ppm S point ppm S point ppm S Temperature (T) 93 . 97** 12 .48** 12. 02** 122. 88** 21.10** 92 . 58* Level (L) 49.65** 343.31** 31.94** 278.48** 106.24** 341.32* T x L 5.22* 6.46* 1.90ms 7.49* 7.08* 137.99' Table 19. Main e f f e c t s of temperature on C0_ compensation and sulphur l e v e l i n leaf tissue. Experiment 8 Barley Peas Rape Temperature C O 2 C0 2 C0 2 day/night Compensation Percent Compensation Percent Compensation Percent °C point ppm S point ppm S point ppm S 18/10 41.7c 0.40a 67.0b 0.35a 82.5a 0.10b 24/16 48.4b 0.39a 74.6b 0.24b 73.9b 0.09b 29/21 64.3a 0.24b 83.7a 0.09c 85.0a 0.39a * Each figure i s the mean of 6 r e p l i c a t e s . Figures followed by the same l e t t e r within a species, and measurement are not s i g n i f i c a n t l y d i f f e r e n t at P = 0.05 according to Duncan's new multiple range t e s t . 113 differences between temperature regimes. Lowest compensation point for barley and peas was observed at 18/10 and f o r rape at 24/16°C. Highest concentration of S i n barley and pea l e a f tissue was also observed at 18/10°C whereas i n rape i t was at 29/21°C. Table 20 gives the simple e f f e c t s of S l e v e l and temperature i n respect to CC^ compensation point and percent S i n the l e a f t i s s u e . S i g n i f i c a n t l y higher CC^ compensation points were associated with s i g n i f i c a n t l y lower S concentration i n l e a f tissue at the three temperature regimes i n a l l three species. This r e l a t i o n s h i p was more pronounced i n barley and peas than i n rape. The c o r r e l a t i o n of CC^ compensation point with percent S i n the l e a f tissue of the three species at the three temperature regimes and at two sulphur l e v e l s was studied. Regression analysis r e s u l t s are given i n table 21. Highly s i g n i f i c a n t correlations between CC^ compensation point and temperature regime, CO^ compensation point and percent S i n the tiss u e and between CC^ compensation point and percent S and temperature, were observed for barley and peas. S i g n i f i c a n t c o r r e l a t i o n c o e f f i c i e n t s were not obtained for rape plants. Table 20. E f f e c t of temperature and sulphur n u t r i t i o n on the CO compensation point i n barley, peas and rape. Experiment 8 Barley Peas Rape Temperature Sulphur C0 2 C0 2 C0 2 day/night l e v e l Compensation Percent Compensation Percent Compensation Percent °C (ppm) point ppm S point ppm S point ppm S 18/10 0 50.5a 0.22a 77.4a 0.28b 93.3a 0.05b 64 33.0b 0.57a 56.6b 0.42a 71.3b 0.15a 24/16 0 56.5a 0.21b 81.3a 0.14b 84.9a 0.06b 64 40.3b 0.57a 68.0b 0.35a 63.16b 0.12a 29/21 0 66.6a 0.12b 88.2a 0.03b 89.2a 0.13b 64 62.1a 0.35a 79.3b 0.15a 80.8b 0.64a * Each figure i s the mean of 3 r e p l i c a t e s . Figures followed by the same l e t t e r within a species, temperature and CO2 compensation point or percent sulphur are not s i g n i f i c a n t l y d i f f e r e n t at P = 0.05 according to Duncan's new multiple range t e s t . t-1 -P 115 Table 21. R squared values from regression analysis (C0 2 being dependent v a r i a b l e ) . Experiment 8 Percent S Species Temperature Percent S + temperature Barley 0.5811** 0.6211** 0.6931** Peas 0.3724** 0.6869** 0.6931** Rape 0.0059ns 0.0081ns 0.0338ns 116 DISCUSSION Development of Sulphur Deficiency Symptoms i n the Three Species The appearance of S deficiency symptoms i n the three species varied i n time of occurrence and l o c a t i o n on the shoot. Earliness of the development of S deficiency symptoms depended on the amount of S i n the seed, i f no S was supplied externally. Although the concentration of S i n rape seeds was greater than i n the other two species,the difference was not s u f f i c i e n t to compensate for the smaller size of the seed. Barley.seeds had the lowest S content compared to the other two species due to t h e i r low S concentration and seed size r e l a t i v e to peas. Peas on the other hand contained the largest amount due to a f a i r l y high S concentration and r e l a t i v e l y large seed s i z e . Peas were able to grow f o r about three weeks before deficiency symptoms developed. Barley and rape developed S deficiency symptoms about two weeks of sowing. The appearance of S deficiency symptoms f o r the three species did not follow the same pattern as Eaton (1966 and Nightingale et al_. (1932 ) reported for avacado and tomatoes. In barley plants, the new growth was evidently at the expense of some S i n the older leaves and an immediate r e d i s t r i b u t i o n of S apparently occurred. This p a r t i a l r e d i s t r i b u t i o n or almost simultaneous d i l u t i o n e f f e c t may have been the cause of c h l o r o t i c symptoms i n the whole plant (Smith, 1962). 117 In rape plants t r a n s l o c a t i o n of S from the older leaves to the younger shoots almost completely depleted the former. As a r e s u l t the new shoot was maintained i n a r e l a t i v e l y healthy state f o r a period u n t i l S deficiency became acute. Biddulph e_t a l . (1958) reported t r a n s l o c a t i o n of S from the older leaves to the younger leaves i n bean plants within 6 35 hours of feeding S . Panak and Szafranek (1967) also reported high concentration of S i n the young rape leaves compared to old leaves and a s i m i l a r d i s t r i b u t i o n i n bean plants. In contrast S i n pea plants had not moved from the older leaves to the younger leaves i n s u f f i c i e n t amount to prevent deficiency symptoms i n younger leaves. Biswas and Sen 3 5 (1959) using S also observed high a c t i v i t y i n the lower leaves of 10 day old pea plants compared with upper leaves. Similar observations were reported by Chapman and Brown (1941) f o r naval orange shoots and Storey and Leach (193 3) f o r tea plants. It i s therefore possible that eit h e r S was withdrawn by proteolysis from old tiss u e and reused i n new tissue as reported by Night-ingale et_ a l . (19 32) or that a protein r i c h i n S was broken down and a protein poor i n S was synthesized under d e f i c i e n t conditions as suggested by Kylin (1953) for wheat plants. The speed of these processes depended on the species as shown by the present observation. I f , as Tompkins et a l . (196 5) reported f o r b r o c c o l i , S d e f i c i e n t plants are supplied with adequate S within about three weeks of planting the severity of the symptoms can be 118 reduced i n 4 to 5 days. This e f f e c t was observed for rape plants i n the present i n v e s t i g a t i o n . Growth Responses to Sulphur N u t r i t i o n Numerous investigators have reported the b e n e f i c i a l e f f e c t s of S on the growth of plants. Similar r e s u l t s were found i n the present i n v e s t i g a t i o n . The fresh and dry weights, shoot length, number of nodes and number of f e r t i l e f r u i t were increased i n a l l three species i f S was adequately supplied. Shoots were affected more by S deficiency than the roots. U l r i c h et a l . (1967) reported that a low l e v e l of S i n a l f a l f a caused the development of root systems proportionately l a r g e r than the shoot systems. Stewart and Porter (1969) reported s i m i l a r r e s u l t s for white clover, wheat and bean plants. The present re s u l t s are i n agreement with the f i n d i n g of these previous workers. For example, the shoot:root r a t i o f o r 0 and 64 ppm S l e v e l at 18/10 were 4.0 and 4.7 f o r barley, 2.1 and 2.8 f o r peas and 1.4 and 2.1 for rape. The increase i n shoot weight with increased S was greater than root weight i n d i c a t i n g that shoot growth was more sen s i t i v e to S l e v e l s than root growth i n the three species. In other words with the onset of S deficiency shoot growth was more depressed than the root growth. The d i f f e r e n t i a l e f f e c t of S on roots and shoots appears to be of formative type as best i l l u s t r a t e d by the shoot:root r a t i o for dry weight. It would be i n t e r e s t i n g to 119 investigate these morphological responses and to correlate them with the changes i n carbohydrate metabolism known to be induced by S deficiency (Ergle and Eaton, 1951). Furthermore an increased root length was recorded i n the present experiments for barley and peas with S deficiency at a l l temperature regimes excepting peas at 13/10 and 35/27 and barley at 18/10°C. Eckerson (1932) also reported s i m i l a r response for S d e f i c i e n t tomato plants. She found that the lengths of roots increased due to the r e l a t i v e increase of meristamatic a c t i v i t y i n the a p i c a l meristem of roots under S d e f i c i e n t conditions. In contrast rape plants i n the present experiments had shorter roots when S was d e f i c i e n t . I t i s therefore l i k e l y that the root elongation i s i n h i b i t e d under d e f i c i e n t conditions i n these plants and that both shoot and roots are equally affected by S deficiency. Temperature had a d i f f e r e n t i a l e f f e c t on the growth response of the three species. With increase i n the growing temperature from 18/10 to 24/16°C, the dry weights of barley at 64 ppm was increased by 44% at the vegetative stage but at mature stage i t was decreased by 39%. At 0 ppm S l e v e l temperature had l i t t l e e f f e c t , only a s l i g h t increase i n weight with temperature was observed. Pea plants responded to temp-erature i n the same manner as barley plants. Rape plants increased by 116% at the vegetative stage and 20% at the mature stage when grown at 29/21 compared to 18/10°C. As with the other two species temperature e f f e c t at 0 S l e v e l was n e g l i g i b l e . 120 It i s evident from these r e s u l t s that the optimum temperature for the growth of barley and peas i s near 24/16°C at the vegetative stage and 18/10°C at the mature stage. On the other hand the optimum temperature f o r the growth of rape plants did not seem to change with the age of the plant. Most rape v a r i e t i e s are adapted to extremes of temperature since they are known to grow equally well i n both cold and warm regions. Canvin (1965) reported that the best response to f e r t i l i z e r for rape was at 26.5°C. In the case of barley and peas a low growing temperature regime (18/10°C) at the mature stage may have maintained a higher photosynthetic:respiration (P:R) r a t i o than at a higher (24/16) growing temperature regime. This seems reasonable, since at higher temperatures mature plants would have more senescing leaves than at lower temperatures and consequently a smaller P:R r a t i o would be expected. The deleterious e f f e c t of high temperature which increases with age of pea plants was reported by Adedipe (1969). These r e s u l t s f o r peas and barley are also consistent with the findings of Went (1957), St a n f i e l d et a l . (1966), Wang and Bryson (1956), Power et a l . (1963) and Tingle (1968). With increase i n temperature the number of nodes increased which i s i n agreement with the findings of S t a n f i e l d et aJL. (1966). Largest numbers of nodes were produced i n plants grown with the highest l e v e l of S (64 ppm) and at a higher temperature, usually 29/21°C. This trend was observed i n a l l 121 three species. Such a response to temperature could be attr i b u t e d to a permanent morphogenetic change that i s brought about by temperature. I f S i s not l i m i t i n g the e f f e c t of temperature i s enhanced. On the other hand i f S i s l i m i t i n g the plant i s unable to respond f u l l y to the environment and adapt successfully within the realm of i t s morphological f l e x i b i l i t y . The y i e l d of f r u i t of the three species were s i m i l a r to the shoot and root weights with respect to S l e v e l s and temperature. As reported f o r barley (Tingle, 1968) and peas (St a n f i e l d et a l . , 1966 and Wang and Bryson (1956) highest y i e l d s were obtained at 18/10°C. Rape yi e l d s were highest at 24/16°C (Wilson, 1967). However, the l a t t e r species had generally low yie l d s at a l l temperature regimes, probably due to the absence of p o l l i n a t i n g agents under growth chamber conditions. Yields of f r u i t s were evidently a r e f l e c t i o n of the growing conditions both before and a f t e r f r u i t i n g . The e f f e c t of S was more obvious at the optimum growing temperatures than at other temperatures. For example the ear heads of barley grown at 18/10°C were 7.9, 50.5 and 47.3 percent of the t o t a l dry weight of a plant at 0, 8 and 64 ppm S leve l s r e s p e c t i v e l y . On the other hand the ear heads of barley grown at 24/16°C were only 0.39, 36.6 and 39.2 percent of the t o t a l dry weight at 0, 8 and 64 ppm S. 122 It i s therefore l i k e l y that when plants are grown under optimum temperature conditions with adequate S, maximum f r u i t set and maximum translocation of food material from other parts of the plant to the f r u i t take place. This mobilization of food materials has been c l e a r l y demonstrated by several workers (Leopold, 1964-). The mechanism of such mobilization i s not completely understood. E f f e c t of Removal of Cotyledons or Endosperm on Sulphur Responses Removal of cotyledons or endosperm tended to cause an i n i t i a l set back i n the growth of a l l three species, r e s u l t -ing i n considerable masking of the e f f e c t of S l e v e l s . In view of the importance of the carbohydrate supply for metabolic processes, i t i s probable that the removal of cotyledons removes most of t h i s reserve food material and thereby causes a greater reduction i n growth than that due to S deficiency alone. After about 10 days, growth increases were observed with the recovery of photosynthetic capacity due to the growth of new leaves. K y l i n (1953) observed a s i m i l a r e f f e c t on deseeded wheat plants. Barley was only s l i g h t l y affected by the removal of endosperm because part of the food reserve may have already been u t i l i z e d by the seedling before excision. Rape plants were more severely affected due to the removal of cotyledonary leaves which comprise the major photosynthetic tissue of the young plant. Peas were also greatly affected because most of the seed reserve i n the cotyledons had not been used up by the 123 seedlings before removal. It seems therefore that the excision of cotyledons i s not a s a t i s f a c t o r y method to be employed for the study of S deficiency, as the l e v e l of other nutrients and organic compounds i n the plant could be l i m i t i n g . Dijkshoorn and Van Wijk (1967) also reported that when cotyledons from Lupinus albus were removed the organic nitrogen content was lower thereby a f f e c t i n g the level of other organic compounds as well. Mineral Concentration and Uptake Generally a higher mineral concentration at the vegetative stage than at the mature stage was observed f o r pea plants p a r t i c u l a r l y at the higher S l e v e l (64 ppm). For example, r a t i o s f o r the two stages (vegetative/mature) f o r t o t a l - S , SQ^-S and t o t a l N at 64 ppm were 2.8, 1.4 and 3.3 re s p e c t i v e l y . These concentrations i n barley tended to remain the same fo r both stages, except f o r total-N which decreased with maturity. The mineral concentrations i n rape plants also decreased with age. Generally f r u i t s , p a r t i c u l a r l y the seeds, are able to accumulate S and N from the vegetative parts of the plant. 3 5 Thomas et a l . (1944) reported 60 to 80 percent S translocation to the f r u i t s during ripening. Petrie et a l . (193 9) reported that as much as 85 percent of the N and 90 percent of the P i n the leaves of wheat for example were retranslocated to the seeds. Due to t h i s enhanced translocation to f r u i t s a f a i r l y consistent 124 SO^-S l e v e l was maintained i n the f r u i t s i n the present experiments even when grown at various temperature regimes. For example, barley grown with 64 ppm S l e v e l at 18/10, 24/16 and 29/2l°C had 0.092, 0.097 and 0.092 percent SO^-S concentration i n the pods. The r e s u l t s for peas and rape were s i m i l a r . Usually even under adverse conditions of growth the n u t r i t i o n a l l e v e l s of the reproductive parts of plants are maintained at the expense of other plant t i s s u e s . I f there i s an acute deficiency of an element such as S, tran s l o c a t i o n to the reproductive parts w i l l be l i m i t e d as i s evident from the data f o r 0 ppm S l e v e l s . Harrison e_t al_. (1944) reported that there i s a v a r i a t i o n of S concentration between d i f f e r e n t structures of the f r u i t . Some parts such as seeds have an a b i l i t y to accumulate more S than other parts such as the glumes and awns of barley seed. Due to a combined analysis of a l l these struc-tures of the f r u i t i n the present i n v e s t i g a t i o n the concentration of SO^-S and t o t a l N tended to be s l i g h t l y lower than i n the other parts of the-shoot. This could be at t r i b u t e d to a d i l u t i o n e f f e c t . Total S concentration i n the shoot tissue of barley and peas increased with increase i n the supply of S. For example, at the mature stage barley shoot tissue had 0.05, 0.07 and 0.34 percent t o t a l S for 0, 8 and 64 ppm S l e v e l s when grown at 18/10 °C. S i m i l a r l y peas had 0.05, 0.05 and 0.11 percent, while rape 125 had 0.08, 0.15 and 0.61 percent f o r the same le v e l s and temperature regime. This i l l u s t r a t e s the fact that the concentration of S i n the tissu e depends on the amount available to the plant. With increase i n temperature there was an increase i n the S uptake at the higher S l e v e l s (8 and 64 ppm) f o r the three species. Barley at 64 ppm contained 1.8, 2.3 and 2.8 mg/g of shoot tissue at 18/10, 24/21 and 29/21°C res p e c t i v e l y . Peas and rape plants had a s i m i l a r response. The values were 0.58, 0.90 and 1.1 for peas and 3.8, 6.3 and 6.5 for rape for the same temperature regimes re s p e c t i v e l y . As Mckell and Wilson (1963) pointed out, higher temperatures do not appear to aid i n S uptake, i f the a v a i l a b l e S l e v e l i s too low. The present experiments showed that the temperature e f f e c t on S uptake becomes apparent i f the l e v e l i s increased from 0 to 8 ppm. Raj an (196 8) reported that the i n i t i a l rapid uptake of SO^ by roots i s a d i f f u s i o n phenomenon, and i s f i r s t accum-ulated i n the apparent free space (AFS) of the root (Epstein, 1955, 1960, and Hylmo, 1958) and i s transported to the xylem by an active process (Brouwer, 1959). Kylin and Hylmo (1957) reported that both "passive" and "active" processes are important i n SO^ uptake by roots. They observed that at low S0 U concentrations the amounts taken up by the metabolic processes 126 w i l l exceed those involved i n physical uptake, whereas at high SO^ concentrations the reverse e f f e c t could be expected. It i s therefore possible that at the highest l e v e l of S (64 ppm) in the present experiments SO^ may have been taken up by a passive process, while at the lower l e v e l s of S (0 and 8 ppm) metabolic energy requiring ("active") processes may have been operating and temperature increases probably had greater e f f e c t . McKell and Wilson (1963) suggested that the increase i n S uptake i n the tops and roots of clover plants as the temperature increased may have been caused by temperature acceleration of the process of ion accumulation. They a t t r i b u t e the movement of S from roots to tops to increased t r a n s p i r a t i o n . Hylmo (1953) and Thomas et a l . (1950) also stressed the importance of t r a n s p i r a t i o n i n S tr a n s l o c a t i o n . The optimum temperature f o r the maximum uptake of S was dependent on the growth stage. For instance barley had the maximum uptake at 24/16 and 18/10 for vegetative and mature stages of growth r e s p e c t i v e l y . Pea plants had the maximum uptake at 29/21 and 18/10°C at vegetative and mature stages. Although the t o t a l S and SO^-S concentrations increased with temperature i n barley and peas, the t o t a l uptake was highest at 18/10°C. This could be attr i b u t e d to better growth with increase i n dry matter at or near optimum temperatures than at high temperatures. 127 The data f o r rape plants indicate that they were better adapted to higher temperatures than the other two species. Both concentrations and t o t a l uptake i n rape increased with increase i n temperature from 18/10 to 29/21°C. The amount of SO^-S taken up by the plant and the amount incorporated into organic compounds depend on the supply and l e v e l of other nutrients. If the supply i s inadequate or barely adequate, p r a c t i c a l l y a l l the sulphur taken up w i l l be incorporated into organic form and l i t t l e or no SO^ w i l l remain (Thomas et_ a l . , 1950 ; Dijkshoorn and Van Wijk, 1957). The present experiments showed that smaller values for SO^-S concentration occur i n S d e f i c i e n t plants. For example the accumulation of SO^-S at 0 l e v e l for barley grown at 18/10°C was 17.5 times lower than at 64 ppm. These r a t i o s did not maintain the same magnitude of difference f o r d i f f e r e n t temperature regimes. At 29/21°C SO^-S i n barley at 0 l e v e l was 10.8 times lower than at 64 ppm. S i m i l a r l y the SO^-S concentration i n plants grown at 8 ppm was 20.2 times lower at 18/10 than at 29/21°C. Even at such low l e v e l s SO^-S concentration increased with temperature. Therefore, when SO^-S l e v e l i s used for the purpose of diagnosing S deficiency i n plants as suggested by U l r i c h et_ a l . (19 59) and Walker and Bentley (1961) i t w i l l be necessary to supply more information such as the growing conditions p a r t i c u l a r l y temperature (Ensminger and Freney, 1965). I f the c r i t e r i o n of 128 " c r i t i c a l percentage" i s used to diagnose sulphur deficiency as U l r i c h and Hylton (1968) suggested, 840 ppm S obtained from barley tissue grown with 8 ppm S at 29/21 would be much higher (100 ppm " c r i t i c a l percentage" for b a r l e y ) , than 130 ppm obtained from barley tissue grown with 8 ppm at 18/10°C, although i n actual fact the best growth with maximum t o t a l uptake of S was obtained at 18/10°C. The concentration of t o t a l N i n the shoot tissue of the three species was s l i g h t l y higher i n plants r e c e i v i n g S at a l l temperature regimes during the vegetative stage of growth. However, at maturity highest concentrations were recorded i n S d e f i c i e n t plants. The t o t a l N concentration at 0 and 64 ppm S l e v e l s i n barley, peas and rape at 18/10°C were 2.87 and 1.79, 4.64 and 1.42, and 3.78 and 3.52 percent r e s p e c t i v e l y . These r e s u l t s are i n agreement with the findings of Tisdale e_t a l . (1950) who also reported high t o t a l nitrogen concentration i n S d e f i c i e n t a l f a l f a . Increases i n t o t a l N concentration are due to accumulation of n i t r a t e nitrogen, amide nitrogen and free amino acids associated with s l i g h t increases i n dry matter. The present r e s u l t s therefore confirm the findings of the other workers. The practice of using crude protein (Total N x 6.25) as a c r i t e r i o n of crop q u a l i t y i s not a r e l i a b l e method. Usually during the f i r s t few weeks S i s not l i m i t i n g due to the presence of S i n the seed and possibly a small percentage from the atmosphere i s also available to the plants (Seim et a l . , 129 1968). Under such conditions protein synthesis would proceed normally maintaining a proper N:S r a t i o which i s close to 15:1 (Stewart, 1969). It i s probably for t h i s reason that N concentration did not increase during the f i r s t 4 weeks of growth. The increase i n N concentration i n S d e f i c i e n t plants at the l a t t e r stages may therefore be the r e s u l t of l i m i t e d protein synthesis. Although there were no temperature e f f e c t s on t o t a l N concentration, t o t a l uptake of N was found to increase with increase i n temperature. These responses were somewhat p a r a l l e l to those of t o t a l S uptake, yet differences among le v e l s within temperatures did not have the same magnitude as for S. This indicates that although the t o t a l uptake of N was lower at low S l e v e l s the t o t a l N concentration i n the tissue was high enough to reduce the differences between l e v e l s . Generally temperature was not as important to N uptake as i t was to S uptake. However, S l e v e l i s important to N uptake as previously noted. NOg concentration i n the tissue of the three species at d i f f e r e n t temperatures showved i n t e r e s t i n g trends. For example, barley grown at 18/10°C had the l e a s t NO^-N concentration of a l l three temperature regimes at a l l three S l e v e l s . S i m i l a r l y peas also had the lowest concentration at the lowest temperature regime (18/10°C). 130 On the other hand at 29/21°C a l l three species at the three l e v e l s had the highest concentration excepting i n rape at 64 ppm l e v e l which had a lower concentration. In view of these findings i t i s l i k e l y that temperature may have had an e f f e c t on NO.-N concentration i n the three species. Eppendorfer (1968) also found low N03-N concentration i n barley at 18°C growing temperature and at high l i g h t i n t e n s i t y . Nightingale et a l . (1932) and Eaton (1941) suggested that n i t r a t e reductase a c t i v i t y i s affected by temperature. Hence i t i s possible that growing temperatures above or below the optimum could reduce the a c t i v i t y of the enzyme and r e s u l t i n an accumualtion of n i t r a t e . Nitrate reductase i s an adaptive enzyme (Bandurski, 1965) and would respond quickly to changes i n the n u t r i t i o n a l status of the plant. When S d e f i c i e n t plants are grown at supra optimal temperatures high concentrations of NO^-N could occur. For example, barley grown at 0 ppm S l e v e l and at 18/10 and 29/21°C temperature regimes had NO^-N concentrations of 0.21 and 0.68 percent res p e c t i v e l y . S i m i l a r l y peas had 0.12 and 0.35 percent fo r the 0 S l e v e l and same temperature regimes. On the other hand rape had lowest values, .33 and .34 percent at 64 ppm S f o r 18/10 and 29/21°C r e s p e c t i v e l y , apparently i n d i c a t i n g that rape plants are better adapted to extreme temperatures (18/10 and 29/21°C) than to intermediate temperature. 131 Since the organic forms of S and N are involved to a large extent i n the synthesis of proteins, the r a t i o of organic N:organic S i n the plant should be close to that i n proteins. Although the amount of t o t a l N and t o t a l S i n the plant changes due to external f a c t o r s , N:S r a t i o s of proteins do not seem to change appreciably (Stewart and Porter, 1961; Stewart, 1969; Dijkshoorn and Van Wijk, 1967). Pumphrey and Moore (1965) also found t h i s r a t i o to be u s e f u l , yet Walker and Bentley (1961) pointed out that i t i s not a precise i n d i c a t o r of S deficiency. Total N:total S r a t i o s were calculated from the data i n the present experiments and indicated that when adequate S i s supplied to plants N:S r a t i o s tend to be smaller i n d i c a t i n g normal synthesis of protein without accumulation of non-protein-N (Dijkshoorn and Van Wijk, 1967 ). On the other hand,, at low l e v e l s of S, N:S r a t i o s were greatly increased due to the increase i n t o t a l N probably consisting mainly of non-protein N f r a c t i o n s (Adams and Sheard, 1966). Ratios obtained f o r barley at 18/10°C were 61, 20 and 5, for 0, 8 and 64 ppm l e v e l s , r e s p e c t i v e l y . S i m i l a r l y peas and rape had 89, 27, 12 and 45, 15 and 6 for the three l e v e l s i n the same order. According to Dijkshoorn and Van Wijk (1967) and Stewart and Porter (1969) N:S r a t i o s below 14 to 1 f o r grasses and 17 to 1 f o r legumes indicate an adequate amount of S i s available i n the plant. I f these recommendations are d i r e c t l y applied to the data, only the 64 ppm S l e v e l could be considered adequate f o r the three 132 species. In other words both 0 and 8 ppm are inadequate f o r normal growth. Increases i n temperature increased the S concentration i n barley tissue at 0 and 8 ppm S l e v e l s . Hence the N:S r a t i o s i n barley were considerably reduced at higher temperatures. At 64 ppm S concentration i n barley tiss u e was low at the high temperature regimes (24/16 and 29/21°C) thereby increasing the N:S r a t i o s . The temperature e f f e c t on r a t i o s i n pea plants at 8 ppm and 64 ppm was small, yet at 0 ppm generally large r a t i o s were recorded at high temperature regimes mainly as a r e s u l t of decreasing S concentrations. In 4 week old pea plants S concentration at 0 l e v e l was increasing with increasing temperature. With N concentration being about the same the r a t i o s tended to be smaller. This i l l u s t r a t e s the importance of stage of growth and other factors such as temperature when using N:S r a t i o s for the purpose of diagnosing S d e f i c i e n c y . The following data further support t h i s observation. Mature pea plants at 0 ppm S l e v e l had t o t a l N:S r a t i o s of 89 and 66 at 18/10 and 24/16°C whereas the 4 weeks old plants had 37 and 34 at the same temperature regimes and S l e v e l . This difference due to age could be attributed to the a v a i l a b i l i t y of a small amount of S i n the seeds. Afte r a period of growth t h i s supply was depleted and the concentration i n the tissue tended to decrease r a p i d l y r e s u l t i n g i n a high N:S r a t i o . 133 Rape plants behaved i n an e n t i r e l y opposite manner to barley. The N:S r a t i o s at higher S l e v e l s decreased as temperature increased due to increase i n S concentration and decrease i n nitrogen concentration. At low l e v e l s S concentration decreased with increase i n temperature but N concentration was maintained at the same l e v e l . This probably was due to a r e l a t i v e increase of non-protein-N as a r e s u l t of S deficiency (Adams and Sheard, 1966 ; Tisdale et a l . , 1950). The present experiments thus indicated two problems i n using t o t a l N:total S r a t i o s f o r diagnostic purposes. F i r s t l y , the growing environmental conditions should be taken into account. For instance, barley plants grown at 18/10°C with 8 ppm S l e v e l had a N:S r a t i o of 19.8 and when they were grown at 2 9/21°C the N:S r a t i o was 17.7. I f the generally recommended N:S r a t i o of 15 i s employed, then 8 ppm l e v e l at 29/21°C seems almost adequate, when i n fact the best growth with highest S uptake was obtained at 18/10°C. Therefore the N:S r a t i o was modified by temperature. Secondly, the stage of growth should be taken into consideration as was suggested by Ensminger and Freney (1966). Data f o r the stages of growth d i f f e r e d considerably. In other words a S l e v e l that i s adequate at H weeks would be i n s u f f i c i e n t at mature stage. In view of the above observations i t i s more r e l i a b l e to use the protein N:protein S r a t i o instead of t o t a l N:total S 134 r a t i o for the purpose of diagnosing S deficiency as Dijkshoorn and Van Wijk (1967) and Stewart and Porter (1969) indicated. On the other hand, t o t a l N:total S r a t i o s could be employed for diagnostic purposes provided they are supported by add i t i o n a l information such as dry weights. Photosynthesis Generally the net C0 2 exchange rates were found to be greater at 20 days than at 30 days, at a l l temperatures excepting the 18/10°C growing temperature regime i n barley and rape. This difference was more pronounced i n peas than i n the other two species. Decline i n the net C0 2 exchange rates with age was even greater under S d e f i c i e n t conditions than under normal conditions.- For example, at the 18/10°C growing temperature regime, the net C0 2 exchange rate i n 30 day old S d e f i c i e n t pea plants was only 33 percent of the exchange rates at 20 days. S m i l l i e (1962) reported a c o r r e l a t i o n between photosynthetic rate of growing pea leaves and the a c t i v i t i e s of photosynthetic enzymes. A s i m i l a r c o r r e l a t i o n for r e s p i r a t o r y rates and several r e s p i r a t o r y enzymes was also found. He concluded that with increasing p h y s i o l o g i c a l age of leaves the enzyme a c t i v i t i e s are reduced, and consequently both r e s p i r a t i o n and photosynthesis are reduced. Trebarne et a l . (1968) also reported for Dactylis  glomerata that the maximum rate of photosynthesis was maintained 135 for 15 to 20 days a f t e r f u l l l e a f expansion. Azmi (1959) reported a decline i n net C0 2 exchange rates at 8 weeks to 2 2 percent of the 2 week rate i n r i c e plants grown at 3 5/18°C. Therefore with increasing age of the plant (Leopold, 1954) a decline i n the photosynthetic rate could be expected. Williams (1936) found that when minerals are inadequate for plant growth, l e a f senescence i s hastened. S i m i l a r l y i f S i s l i m i t i n g , S d e f i c i e n t leaves would probably senesce much sooner than normal leaves. A l t e r n a t i v e l y a decline i n C0 2 exchange rates i n S d e f i c i e n t leaves could be due to mesophyll resistance to C0 2 exchange as was observed for N d e f i c i e n t cotton and bean leaves by Ryle and Hesketh (1968). They found that stressing the leaves f o r N increased the mesophyll resistance, through e f f e c t s on photochemical and biochemical reactions of photosynthesis and r e s p i r a t i o n . Reductions i n net CG^ exchange rates i n 3 0 day old rape plants due to S deficiency were more pronounced at higher temperatures than at lower temperatures. Consequently greater r a t i o s between the net C0 2 exchange rates at 64 and 0 ppm S l e v e l s were obtained. Similar trends were also observed i n barley and peas. Hence, i n addition to the e f f e c t of p h y s i o l o g i c a l age and hastened senescence, S d e f i c i e n t plants may also have exhausted the avai l a b l e S supply i n the seeds a f t e r 30 days and the deleterious e f f e c t s of temperature would be enhanced. 136 S i g n i f i c a n t differences i n net C0 2 exchange rates due to both growing and measuring temperatures i n respect to S l e v e l s were observed. However, these differences i n rape plants were small at higher temperatures. For example,the r a t i o s between 0 and 64 ppm S le v e l s at 18/10, 24/16, 29/21 and 35/27°C growing temperatures were 1.4, 1.1, 1.1 and 1.0 res p e c t i v e l y . This smaller difference at higher temperatures could be att r i b u t e d to the fact that rape plants are more adapted to higher temperatures than lower temperatures and even under adverse conditions, such as with inadequate S l e v e l s , they are able to maintain the photosynthetic a c t i v i t y at r e l a t i v e l y high rates. Barley and peas had opposite trends with regard to net exchange rates at d i f f e r e n t temperature regimes and S l e v e l s . Ratios f o r barley ranged from 1.5 to 1.8 and f o r peas from 1.1 to 1.3 from the lowest to the highest growing temperature regimes (18/10 to 35/29°C). These two species are normally grown during the cool season. Barley grows at an optimum temperature of around 18°C (Weaver, 1950, Scheibe and Ellerman, 1968 ; Power et al_. , 1964). and has the maximum C0 2 exchange rates between 16 and 21°C (Ormrod et_ a l . , 1968). When these species are subjected to high growing temperature regimes (24/16, 29/21 and 35/27°C) the net C0 2 exchange rates would tend to be low and the magnitude of t h i s e f f e c t would be even greater under S d e f i c i e n t conditions than under normal conditions. Some of the detrimental e f f e c t s of temperature 137 could be overcome because of the presence of adequate S i n the plant. Increase i n measuring temperature above the growing temperature caused no further stimulation i n CC^ uptake, and at higher temperatures there was a decrease i n CC^ uptake. These r e s u l t s agree with the findings of Treharne et a l . (1968) who found that when orchard grass was' grown at 29/21°C (day/night) and measured at 29 and 21°C there was a higher rate of apparent photosynthesis at the former temperature than at the l a t t e r , s i m i l a r l y when i t was grown at 21/13°C there was no increase i n apparent photosynthesis with r i s e ' i n temperature, suggesting that the leaves of orchard grass photosynthesized optimally at or near the temperature at which the leaves developed and grew. Hesketh (1968) also reported that optimum temperatures f o r photosynthesis could change with a change i n external conditions. Excepting for 20 day old rape plants, a l l three species at both S l e v e l s had maximum exchange rates either at the growing temperature or at a lower temperature than the growing temperature. Even those grown at 35/27°C had the maximum net CO^ exchange rate at 35°C or below. As Mooney and West (1964) suggested, both rate modifications and s h i f t s i n temperature of the optimal photosynthesis were observed. It i s not known whether t h i s adaptation i s r e f l e c t e d i n anatomical and morphological characters as El-Sharkawy and Hesketh (1964) and Wilson and Cooper (1969) suggested, 138 i n s i m i l a r studies, or due to some endogenous rhythm as Salisbury and Ross (1969) suggested. The r e s u l t s obtained by Ormrod et al_. (1968) f o r barley also seem to have s i m i l a r responses. In t h e i r experiment barley plants were grown at 24/16°C (day/night) and measured at a range of temperatures between 4 and 34°C. In addition to the v a r i e t a l differences observed i n respect to net exchange, maximum exchange rates i n a l l v a r i e t i e s tested were recorded below 24°C which was the growing temperature. When the CC^ exchange rates are expressed onthe 2 basis of area (dm ) and weight (gdw or gfw) rates d i f f e r e d greatly f o r the two S l e v e l s (0 and 64 ppm). The magnitude of t h i s difference between 0 and 64 ppm S l e v e l s was greatest when expressed on a weight basis. Usually CG^ exchange rates are reported on an area basis, although expression on a weight basis i s not uncommon. Large differences observed on a weight basis between the S l e v e l s may be at t r i b u t e d to lack of c o r r e l a t i o n between leaf area and le a f weight i n S de f i c i e n t leaves. Nightingale (1932) reported that under S d e f i c i e n t conditions plants accumulate large amounts of carbohydrates, mainly due to impaired protein synthesis. I t i s therefore l i k e l y that S d e f i c i e n t leaves are heavier than normal leaves on a per unit leaf area basis. Barnes et_ a l . 139 (1969) reported that the s p e c i f i c l e a f weight (SLW = le a f dry weight per unit l e a f area) of a l f a l f a changed with va r i e t y and growing conditions. Pearce et a l . (1969) found a p o s i t i v e c o r r e l a t i o n between SLW and net photosynthesis i n a l f a l f a . Murata's (19 61) observation that high l e a f area:leaf weight r a t i o s i n r i c e were associated with low photosynthetic rates also support t h i s observation. In contrast, although the SLW of S d e f i c i e n t leaves would be larg e r , the net photo-synthesis was smaller. Hence i t i s possible to obtain exaggerated differences i n net CC^ exchange rates i n plants grown at d i f f e r e n t S l e v e l s . In addition to actual rate differences due to S deficiency, a difference due to lack of c o r r e l a t i o n between area and weight of leaves would also be present as evident from the data of the present experiments. CO,, Compensation Point The fact that some plant species have a higher C O 2 compensation point than others has been generally accepted. Tregunna and Downton (1967) reported a C O 2 compensation of about 50 ppm for some temperate species, and 0-5 ppm f o r some t r o p i c a l species. Hofstra and Hesketh (1969) have reported various c h a r a c t e r i s t i c s which d i f f e r between species with and without photorespiration. Of the two groups the former i s 140 known to have the Calvin carboxylation reaction and the l a t t e r the C-4 pathway or phosphopyruvate carboxylation reaction (Hatch and Slack, 1966; Hatch et a l . , 1966 and Slack and Hatch, 1967). Barley, peas and rape plants used i n the present i n v e s t i g a t i o n belong to the group that has a high CO^ compensation point and the values obtained are generally i n agreement with those reported i n the l i t e r a t u r e . For example the three species grown with adequate S (64 ppm) had C O 2 compensation values of 3 3 and 51 ppm for barley and peas respectively at 18/10°C, and 63 for rape at 24/16°C. On the other hand when S was l i m i t i n g , barley and peas had C O 2 compensation values of 50 and 77 ppm resp e c t i v e l y at 18/10°C and rape had 8 5 ppm at 24/16°C. Since plants with higher C O 2 compensation points f i x smaller amounts of C O 2 than those with lower compensation points the photosynthetic rates and dry weights of S d e f i c i e n t plants were accordingly smaller as evident i n other data from the present experiments. Evidence i s mounting to suggest that r e s p i r a t i o n i n the l i g h t i s d i f f e r e n t from that i n the dark (Decker, 19 55; Tregunna et a l . , 1961, 1964; Forrester et a l . , 1966). Those plants with high C O 2 compensation points are known to have r e s p i r a t i o n i n the l i g h t (photorespiration) and that due to such r e s p i r a t i o n apparent photosynthetic rates are low. Hofstra and Hesketh (1968) found that the maximum rates of photosynthesis and photorespiration occur at about the same 141 temperature. However, the temperature f o r maximum rate for dark r e s p i r a t i o n was found to be 10°C higher. In the present in v e s t i g a t i o n when the growing temperatures of 18/10 and 24/16°C were compared for barley and peas the CO^ compensation value was higher f o r the higher growing temperature, when a l l measurements were taken at 21°C. This increase was greater i n plants grown with adequate S than under S d e f i c i e n t conditions. Rape plants, on the other hand, had higher values, only when the growing temperature was 29/21 rather than 18/10° or 24/16°C. The increase i n the CC^ compensation point of normal plants with increase i n temperature could probably be due to an increase i n photorespiration or decrease i n photosynthesis or both ( J o l l i f f e and Tregunna, 1968). In the present experiments the change i n the CO^ compensation point was due to the growing temperatures since a l l measurements were taken at 22°C. 14 Yudina e_t a l . (1969), using glucose C on pea Sprouts, determined the Cg/C^ r a t i o at 25 and 3 8°C temperatures. They found that the high temperature increased the rate of resp i r a t o r y metabolism, but did not change the r e l a t i o n between the g l y c o l y t i c and pentose phosphate pathways. It i s therefore l i k e l y that an increase i n photorespiration or dark r e s p i r a t i o n (Graham and Walker, 196 2) and not a change i n the pathway of r e s p i r a t i o n was the cause of the higher C0 o 142 compensation point i n normal (64 ppm) plants at increased temperature regimes. The r e s u l t s of the c o r r e l a t i o n study show that C0 2 compensation points of barley and peas are p o s i t i v e l y correlated with temperature and negatively correlated with S l e v e l . In other words a decrease i n S would r a i s e the compensation point, and an increase i n temperature also would have s i m i l a r e f f e c t s . If both high temperature and low S l e v e l s are present the e f f e c t on the C0 2 compensation point would be more pronounced. El-Sharkawy and Hesketh (196 5) have reported that C0 2 a s s i m i l a t i o n rate i n plants with photorespiration i s smaller than those with no photorespiration. Therefore, i n order to obtain higher y i e l d s these plants should be grown at a low optimum temperature regime of around 18/10°C (Ormrod et a l . , 1968 , Tingle, 1968), with adequate S l e v e l s (Thomas et a l . , 1943 ; Thomas, 1965). It i s not c l e a r why the rape plants did not have the same c o r r e l a t i o n s . Perhaps the d i f f e r -ence i s due to a d i f f e r e n t temperature response of t h i s species. The e f f e c t of S l e v e l s on C0 2 compensation point has not been studied. Hence, only a few speculations can be stated i n the l i g h t of the present information. S d e f i c i e n t plants were c h l o r o t i c i n nature and s i m i l a r i n appearance to N d e f i c i e n t plants (Maraby, 1968). Tregunna et al.,(1964) grew soya bean, peperomia and corn plants under mineral deficiency conditions and measured t h e i r photorespiration rates. 143 They observed that chlorophyll-poor (mineral d e f i c i e n t ) leaves had no photorespiration. On the other hand, Ryle and Hesketh (1969) reported that photorespiration i s less affected by nitrogen deficiency than i s photosynthesis suggesting that the rate of photorespiration i s not always c l o s e l y linked with that of photosynthesis. This descrepancy could be due to the fact that i n the former case no nutrients were applied at a l l and i n the l a t t e r case only N was l i m i t i n g . Therefore, i f the assumption i s made that S d e f i c i e n t leaves do not have photorespiration, then the increase i n the C O 2 compensation point could be attributed to another process or processes associated with low photosynthetic rate or high C 0 2 evolution or both. There i s some evidence to indicate that the c i t r a t e cycle may be i n h i b i t e d d i r e c t l y or i n d i r e c t l y and that d i r e c t oxidation may dominate i n S d e f i c i e n t plants (Willenbrink, 1966). S i m i l a r l y Goodman et a l . (1967) suggested that i n diseased wheat leaves NADPH2 produced i n the pentose phosphate pathway, may be d i r e c t l y oxidized by a noncytochrome system such as the ascorbic acid oxidase or polyphenol oxidase systems. When t h i s system i s operative i n leaves i t i s associated with higher rates of C 0 2 production (Goodman e_t a l . , 1967). This would r e s u l t i n higher C 0 2 compensation points. I f .the metabolism i n S d e f i c i e n t leaves i s s i m i l a r to diseased leaves, such a type of oxidation may be responsible for the higher C 0 _ compensation points found. 144 SUMMARY AND CONCLUSIONS The influences of 5 temperature regimes and 6 S le v e l s on the growth and mineral composition of barley, peas and rape at vegetative and mature stages of growth were investigated i n controlled environment experiments. Net C O 2 exchange rates were also compared at 4 growing temper-atures and 6 measuring temperatures. Further, the influence of 3 temperature regimes and 2 S l e v e l s on the C O 2 compensation point was studied. From the r e s u l t s of the experiments the following conclusions were drawn. 1. The appearance of S deficiency symptoms did not follow the same pattern i n a l l three species. In barley the whole plant showed c h l o r q t i c symptoms, probably due to continuous r e d i s t r i b u t i o n of S. Pea.plants showed l i t t l e r e d i s t r i b u t i o n u n t i l the l a t t e r stages of growth, while rape plants showed an almost immediate tr a n s l o c a t i o n to the younger tissue from older t i s s u e . 2 . I f S i s not supplied e x t e r n a l l y , the earliness of the development of'S deficiency symptoms depended on the amount of S i n the seed. Barley and rape plants developed symptoms at about 2 weeks and pea plants at about 3 weeks. Also, the lowest S l e v e l treatment at the highest temper-ature regime took the shortest time to develop deficiency symptoms. 145 3. I f S was supplied to S d e f i c i e n t plants a f t e r 2 to 3 weeks, they tended to resume normal growth i n 4 to 5 days, with the disappearance of deficiency symptoms. 4. Shoot and root weights were increased at the highest S l e v e l (64 ppm) i n a l l three species. Greatest response to S during the vegetative stage occurred at 24/16 f o r barley and peas and at 29/21°C fo r rape. At the mature stage the highest response was at 18/10 f o r barley and peas and 29/21°C f o r rape. Yields of f r u i t of barley and peas also increased at 18/10 and rape at 24/16°C. 5. Plants from a l l three species grown at the lower temperature regime (18/10°C) with the lower S l e v e l (0 ppm) had fewer nodes compared to those grown at higher temperature (29/21°C) with a higher l e v e l of S (64 ppm). 6. Shoot and root lengths decreased progressively with increase i n temperature i n a l l three species. Plants with the lower S l e v e l (0 ppm) had greater root lengths i n a l l three species. 7. Removal of cotyledons or endosperm tended to cause an i n i t i a l set back i n the growth of the three species. Barley was s l i g h t l y less affected as part of the food reserve had already been u t i l i z e d before excision. As the detrimental e f f e c t of removal of cotyledon or endosperm was greater than the e f f e c t s of temperature or S l e v e l s , t h i s method was found to be unsatisfactory f o r S n u t r i t i o n studies. 146 8. Mineral concentrations i n the tissue of peas and rape plants were greater at the vegetative stage than at the mature stage. Mineral concentration i n barley tended to remain the same for both stages. 9. When adequate S was availa b l e i n the shoots, f r u i t was able to maintain a constant S l e v e l i r r e s p e c t i v e of growing temperatures, probably due to greater trans-l o c a t i o n from the other parts of the plant to the f r u i t . 10. The optimum temperature f o r maximum uptake of S depended on the growth stage. Barley had maximum uptake at 24/16 and 18/10°C fo r the vegetative and mature stages respect-i v e l y . Peas had maximum uptake at 29/21 and 18/10°C for vegetative and mature stages. Rape had increased t o t a l uptake at 29/21°C at both stages of growth. 11. The increases i n S concentration at higher temperatures were l a r g e l y due to "concentration e f f e c t s " r e s u l t i n g from smaller plants. Therefore SO^-S l e v e l i s not considered a good c r i t e r i o n f o r the diagnosis of S deficiency i n plants. 12. Generally, t o t a l N concentration was higher at lower S lev e l s i n a l l three species. N concentration did not increase during the f i r s t 4 weeks of growth as normal protein synthesis may have been operative due to the presence of S from the seed reserve. Temperature had l i t t l e e f f e c t on t o t a l N uptake. 14 7 13. NO„-N concentration increased at low S le v e l s and at higher temperature regimes. It appears that n i t r a t e reductase a c t i v i t y i s reduced under these conditions r e s u l t i n g i n greater accumulation of NO^-Nl 14. The present experiments indicated two problems with regard to the use of t o t a l N:total S r a t i o s f o r diagnostic purposes: (a) The importance of growing environment, p a r t i c u l a r l y temperature, because S concentration at a p a r t i c u l a r l e v e l changes with temperature. (b) The importance of the stage of growth of plants. Total N:total S r a t i o s at 4 weeks at low S l e v e l s tended to be smaller than at maturity. 15. Generally greater net CO^ exchange rates were found at 2 0 days than at 3 0 days, and were more pronounced i n S d e f i c i e n t plants. This decline i n rates appeared to be due to an increased proportion of p h y s i o l o g i c a l l y aged leaves to photosynthetically active leaves. For example, at the 18/10°C growing temperature regimes, the net CO^ exchange rates i n 3 0 day old S d e f i c i e n t peas were only 3 3 percent of the exchange rates at 2 0 days. 16. Reduction i n net CO^ exchange rates due to S deficiency at higher temperatures was less i n rape than i n barley and peas , i n d i c a t i n g that rape i s more adopted to higher temperatures than the other two species. 148 17. Increase i n measuring temperatures above the growing temperatures caused no further stimulation i n CC^ uptake, and with increased temperatures, there was a decrease i n CC^ uptake. The maximum net CC^ exchange rate was at or below the growing temperature at both S l e v e l s (0 and 64 ppm). 18. Expression of CC^ exchange rates on the basis of l e a f 2 area (dm ) was less variable than on the basis of l e a f weight (gdw). S d e f i c i e n t leaves tended to be heavier (probably due to carbohydrate accumulation) per unit area than normal leaves. 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Appendix I D i a g r a m S h o w i n g R a n d o m A r r a n g e m e n t o f T r e a t m e n t s in E a c h o f t h e T h r e e R o w s in t h e G r o w t h C h a m b e r B: Barley Leve ls : 64 (1), 8 (2) & 0 (3)PPm. PC Peas Replicates: l . l l & l l l . R: Rape Appendix I I . An example of a combined analysis of variance for main e f f e c t s . Source df Sum Sq . Mean Sq. Error F Prob. EXPT e-1 1 43 .0 4 3.0 ROW/ET 0 .14 0.7135 TEMP t-1 . 2 30923 .0 15461.0 EXT 36 .34 0.0374 EXT ( e - D ( t - l ) 2 850 .9 425.5 ROW/ET 1 .38 0.2892 ROW/ET e t ( r - l ) 12 3703 .2 308.6 0 . 86 0.5929 LEVEL A - l 2 187030 .0 9 3514.0 26 . 81 0.0 TXL ( t - l ) U - l ) 4 17251 .0 4312.7 12 .03 0.0 ERROR t ( e r - l ) U - l ) 30 10756 .0 358 .5 TOTAL etr£-l 53 250550 .0 Variable: Total N uptake i n mature barley shoots Temperature: 18/10, 24/16, 29/21 day/night °C Sulphur: 0, 8, 64 ppm r—1 

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