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Some effects of low, non-freezing temperatures on plants Gallopin, Isabel Gomez 1971

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SOME EFFECTS OF LOW, NON-FREEZING TEMPERATURES ON PLANTS by ISABEL CCMEZ GALLOPIN B.Sc. Cornell University, Ithaca, New York, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF •MASTER OF SCIENCE 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 May, 1971 In presenting t h i s thesis in p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for s c h o l a r l y purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or p u b l i c a t i o n o f t h i s thesis f o r f i n a n c i a l gain shall not be allowed without my written permission. Department of The University of B r i t i s h Columbia Vancouver 8, Canada • ii ABSTRACT Low, non-freezing temperatures can cause both, harmful and b e n e f i c i a l effects on plants, and t h i s research was carried out to survey some effects on starch and pigment accumulation. Four species were selected on the basis of photosynthetic biochemistry and major systematic grouping. Zea and Gomphrena possess the C^-dicarboxylic acid pathway t y p i c a l of c e r t a i n fami-l i e s of t r o p i c a l o r i g i n , while Trjticum and Phaseolus contain the Calvin cycle alone which i s t y p i c a l of plants o r i g i n a t i n g i n temperate regions. Zea and Trjticum are Monocotyledoneae while Gomphrena and Phaseolus are members of the Dicotyledoneae. Plants of each species were subjected to 10 days of cold treatment s t a r t i n g when they were 10, 21 or 35 days o l d (15, 26 and HO days o l d f o r Gomphrena), and spectrophotometry measurements of starch, chlorophylls a and b, and carotenoids were carried out during the treatments. The effects of cold temperature depended on species, age, and duration of treatment. A l l of the species exhibited a s i g n i f i c a n t l y higher l e v e l of starch i n the cold temperature f o r at least two of the three ages tested. The most dramatic e f f e c t of low temperature occurred i n Gomphrena when the starch concentration increased to over 2000 per cent of the concentration attained at the warm temperature. Variations i n the e f f e c t of cold treatment between the d i f f e r e n t ages tested were more pronounced i n the monocots used than i n the dicot species studied and variations due to the duration of cold treatment were observed i n Gomphrena and 'Trjticum. Cold treatment also caused s i g n i f i c a n t reduction i n t o t a l chlorophylls, chlorophyll a and chlorophyll b i n a l l the species except Triticum. In Zea, the response to cold decreased as the plants aged, and the duration of cold i i i treatment had a s i g n i f i c a n t e f f e c t i n Zea and Gomphrena. When the youngest plants only are considered, the response of starch and chlorophyll levels to cold treatment was w e l l correlated with the t y p i c a l photosynthetic pathway of the species tested. Low temperature had no s i g n i f i c a n t effect on t o t a l carotenoid concen-t r a t i o n . The e f f e c t of lew temperature on l i g h t transmission by young Zea. leaves during the f i r s t 48 hours of greening was also examined. Chlorophyll a concentration and l e a f l i g h t transmission were highly correlated and the more convenient transmission measurements can therefore be used to predict leaf chlorophyll concentration. At the warm temperature used, there was a l i n e a r increase i n chlorophyll concentration a f t e r a 2 hour lag period. Preceding cold treatment caused a longer lag period before chlorophyll began to accumulate at the warm temperature. Also, no chlorophyll accumulated, or there was net chlorophyll breakdown at low temperature. Kinetin treatment did not prevent the decrease i n chlorophyll concentration at the low temperature. i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES . . . ' v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS ' i x 1. General Introduction •. . 1 2. The Effects of Temperature and Age on Starch and Pigment . Accumulation i n Triticum, Zea, Phaseolus and Gomphrena. Introduction 10 Materials and Methods 11 Results • 19 ( i ) Effects of Temperature and Age on Starch Content . . . 10 ( i i ) Effects of Temperature and Age on Pigment Content. . . 22 a) Triticum vulgare b) Zea mays c) Phaseolus vulgaris d) Gomphrena globosa Discussion 33 3. The Effects of Temperature and Kinetin on the Accumulation of Chlorophyll i n E t i o l a t e d Zea Leaves. Introduction . . . . . . . 40 Materials and Methods . 41 Results and Discussion 42 4. Literature Cited • 52 V Page 5. APPENDIX I. Vertical Gradient i n Light Intensity in the Percival Growth Chamber 59 6. APPENDIX II. Variations i n Chlorophyll Concentration within Plant Leaves 60 7. APPENDIX III. Determination of the Effects of Time, Temperature • . and Light on the Absorbance of Chlorophyll Extracts . . . . . . 62 v i LIST OF TABLES Table Page 2-1 Number of plants used f o r starch determination . . 13 2-2 Fresh weight (gm) of l e a f samples f o r pigment determination. . 15 5- 1 V e r t i c a l gradient i n l i g h t i n t e n s i t y i n the Per c i v a l growth chamber 59 6- 1 Variations i n chlorophyll concentration within plant leaves. . 60 7- 1 Effects of time, temperature and l i g h t on the absorbance of chlorophyll extracts 62 ' LIST OF FIGURES v i i Figure T i t l e Page 2-1 Effe c t of potato starch concentration on the absorbance of aqueous iodine-potassium iodide-starch mixture at 620 nm. . ]_g 2-2 Starch concentrations i n 10, 21 and 35 day-old Triticum (a) and Zea (b) plants subsequently exposed to warm or cold conditions f o r 0, 5 or 10 days 20 2-3 Starch concentrations i n .10, 21 and 35 day-old Phaseolus (a) plants, and 15 and 26 day-old Gomphrena (b) plants" subsequently exposed to warm or^cold conditions f o r 0, 5 or 10 days 21 • 2-4 Total chlorophyll (a) and t o t a l carotenoid (b) concentrations i n 10, 21 and 35 day-old Triticum plants subsequently exposed to warm or cold conditions f o r 0, 5 or 10 days 23 2-5 Chlorophyll a (a) and chlorophyll b (b) concentrations i n 10, 21 and 35 day-old Triticum plants subsequently exposed to warm or-cold conditions f o r 0, 5 or 10 days 24 2-6 Total chlorophyll (a) and t o t a l carotenoid (b) concentrations i n 10, 21 and 35 day-old Zea plants subsequently exposed to warm or cold conditions f o r 0, 5 or 10 days . 26 2-7 Chlorophyll a (a) and chlorophyll b (b) concentrations i n 10, 21 and 35 day-old Zea plants subsequently exposed to warm or cold conditions f o r 0, 5 or 10 days. 27 2-8 Total chlorophyll (a) and t o t a l carotenoid (b) concentrations i n 10, 21 and 35 day-old Phaseolus plants subsequently exposed to warm or cold conditions f o r 0, 5 or 10 days. 29 2-9 Chlorophyll a (a) and chlorophyll b (b) concentrations i n 10, 21 and 35 day-old Phaseolus plants subsequently exposed to warm or cold conditions f o r 0, 5 or 10 days 30 2-10 Total chlorophyll (a) and t o t a l carotenoid (b) concentrations i n 15, 26 and 40 day-old Gomphrena plants subsequently exposed to warm or cold conditions f o r 0, 5 or 10 days 31 2- 11 Chlorophyll a (a) and chlorophyll b (b) concentrations i n 15, 26 and 40 day-old Gomphrena plants subsequently exposed to warm or cold conditions f o r 0, 5 or 10 days 32 3- 1 Changes i n the per cent transmission of l i g h t .through leaves during the i n i t i a l 48 hours of greening of e t i o l a t e d Zea seedlings exposed to warm or cold conditions ..... . . . . 43 VX11 Figure . T i t l e Page 3-2 Variations i n l e a f thickness during the i n i t i a l 48 hours of greening of e t i o l a t e d Zea seedlings exposed to warm or cold conditions 45 3-3 Changes i n chlorophyll a concentration during the i n i t i a l 48 hours of greening of e t i o l a t e d Zea seedlings i n warm con-, di t i o n s ~. 7 46 3-4 Relationship between the per cent transmission of l i g h t through leaves and the leaf chlorophyll a concentration during the greening of et i o l a t e d Zea seedlings i n warm conditions ~ 7 . . 47 3-5 Changes i n the per cent transmission of l i g h t through leaves during the i n i t i a l 24 hours of greening of e t i o l a t e d Zea seedlings 48 3-6 Effe c t of Kinetin on the changes i n per cent transmission of l i g h t through leaves during the i n i t i a l 24 hours of greening of e t i o l a t e d Zea leaves exposed to warm or cold conditions. 51 ACKNOWLEDGEMENTS i x The author wishes to express.her sincere'appreciation f o r the counsel and support given by Dr. P.A. J o l l i f f e throughout these studies. The assistance and advice given by Dr. V.C. Runeckles and Dr. N.R. Bulley i s g r e a t f u l l y acknowledged. Thanks are also extended to Dr. G.W. Eaton f o r the help provided with s t a t i s t i c s and computer programming, to Mrs. M. Hirsekom f o r her technical assistance i n carrying out some of the starch and pigment determinations, to Mr. J . Gibson f o r b u i l d i n g the apparatus used i n l i g h t transmission measure-ments, and to Dr. G.R. L i s t e r from Simon Fraser University who kindly provided the l i g h t f i l t e r s f o r the l e a f l i g h t transmission studies. F i n a l l y , t h i s thesis i s dedicated to my husband, Gilb e r t o , who assisted i n d r a f t i n g the figures and whose encouragement made t h i s thesis possible. 1. GENERAL INTRODUCTION 1 In a comprehensive review of the effects of temperature on plant growth. Went (1953) mentioned that perhaps h a l f of the published papers i n plant physiology could be quoted.since so many contain references to temperature, i f only i n t h e i r description of the experimental conditions. Nevertheless, there i s r e l a t i v e l y l i t t l e information available on the response of plants to low, non-freezing temperatures. Furthermore, i n spite of the fact that the ef f e c t s of temperature are largely mediated by t h e i r e f f e c t s on chemical reactions, i n only some cases has the biochemistry of the plant been studied i n . r e l a t i o n to low, non-freezing temperatures. Nevertheless i n many cases physiological responses to.low non-freezing temperature have been attributed to postulated biochemical changes. * o o The temperature at which most plants grow ranges from 0 C to 40 C. At very low temperatures, reactions can be i n h i b i t e d by an inadequate supply of energy because the lower k i n e t i c energy of the reacting molecules r e s u l t s i n a decreased rate of reaction. At very low and excessively high temperatures, protein denaturation can occur. As a r e s u l t of t h i s , a generalized temperature - response curve f o r the growth rate of plants would be expected to follow the shape of an enzyme temperature - response curve, r i s i n g r a p i d l y at low temperatures, reaching an optimum at intermediate temperatures, and then f a l l i n g at.higher temperatures. Many examples of such responses of plant growth to temperature are known (Evans et a l . , 1964; Went, 1957). I t i s often found that at low temperatures an enzyme reaction possesses an apparent a c t i v a t i o n energy higher than expected (Langridge, 1963). This i s usually at t r i b u t e d to an increase of intermolecular hydrogen bonding. By t h i s hypothesis, such excessive bonding a l t e r s the structure of the enzyme so that the centers of a c t i v i t y lose t h e i r s p e c i f i c configurations, or they are 2 no longer exposed to the substrate (Langridge, 1963). Temperature changes not only d i r e c t l y alter 1 biochemical a c t i v i t y , but can also lead to physical changes i n tissues which can change the equilibrium or rate of an enzymatic reaction. Substrate a v a i l a b i l i t y may be affected by permeability changes which occur at low temperature (Kuiper, 1964).• Also, carbon dioxide s o l u b i l i t y i n water i s higher than that of oxygen at low.temperature, but at higher temperature the reverse occurs. This l a s t e f f e c t may be one reason why the r a t i o of the rates of photosynthesis to re s p i r a t i o n i s so high at low temperatures. As temperature i s increased, respiratory rate i s increased r e l a t i v e l y more than photosynthesis, thus r e s u l t i n g i n lower r a t i o s . This might, explain, at least p a r t l y , why many plants grow better i n temperate regions than i n the tropics (Went, 1953). At low temperatures, the increased amount of carbon dioxide i n the c e l l sap increases i t s a c i d i t y . This w i l l favour carbon dioxide f i x a t i o n i n t o organic acids which w i l l further increase a c i d i t y (Richer et a l . , 194-8). As a consequence of such changes i n enzymatic a c t i v i t i e s , as w e l l as alte r a t i o n s of the physical properties of the c e l l sap, the lowering of temperature may cause changes i n the amount and nature of substrates a v a i l -able to the plant. The increase i n sugar and acids may account f o r some of the observed effects of lowered temperature, e s p e c i a l l y the short term effects on growth. Our knowledge of the effects of temperature on growth has been greatly expanded by the work of Went and h i s collaborators. His f i r s t major con-t r i b u t i o n to t h i s area of knowledge was the discovery that temperatures at night were of s p e c i f i c importance i n the growth of whole plants (Went, 1944). According to species, v a r i e t y , and age of the plant, Went i d e n t i f i e d an 3 optimal night temperature which was d i f f e r e n t from and usually lower than the d a i l y optimum (Went, 1956 and 1957). In Zea, the optimal night temperature depended on the diurnal temperature regime i n which a p a r t i c u l a r plant was maintained. For example, a day temperature of 23° C was associated with a nocturnal optimum of 17° C, but when the day temperature was 30° C, the nocturnal optimum was shif t e d to 20° C. I t i s not ce r t a i n whether the b e n e f i c i a l e f f e c t of lower night temperatures i s due to the absolute values of temperature experienced by the plant or whether the simple experience of some f l u x of temperature i s the b e n e f i c i a l item. A suitable night temperature may also have b e n e f i c i a l effects of plant development-. Trie earliness and i n t e n s i t y of flowering and f r u i t i n g can be affected by night temperature (Went, 1953). For example, i n Lolium perenne, L. perenne: L.X multiflorum, Dactylis glomerata, Agrostis tenuis, Holeus  lanatus, T r i f o l i u m repens, T. subterraneium and Lotus uliginosus, lower day temperature gave a much greater relative reduction i n growth of i n d i v i d u a l t i l l e r s than d i d a corresponding reduction i n night temperature. In contrast, a lowering of the day temperature had a r e l a t i v e l y small influence on the ' rate of t i l l e r i n g of most species, but i n some cases lower night temperatures resulted i n subs t a n t i a l l y higher rates of t i l l e r i n g ( M i t c h e l l , 1955 and 1956). In " c h i l i pepper" plants, the optimum night temperatures f o r stem elongation decreased from 30° C to 8.5° C as the plant progressed to maturity (Dorland, and Went, 1947). Low non-freezing temperatures during the day may also have b e n e f i c i a l e f f e c t s on growth. For example, i n Poa pratensis, growth at 15° C was greater than at 25° and 35° C (Darrow, 1939, Hiesey, 1953). Brdmus carinatus, B. r i g i d u s , B. rubens, M e l l i c a imperfecta, Stipa ' l i p i t a , Poa s c r a b e l l a , a l l grew w e l l at day/night temperatures of 20°/3° or 6° C or 10° C, but t h e i r growth was r e s t r i c t e d at 30°/3° (Ashby and HelTmers, 1959). In many t r o p i c a l plants death i s caused by temperatures w e l l above the freezing point (Went, 1953). This occurs i n Coleus, Saintpaulia, P a l i s o t a and many other genera, and i t i s associated with loss of sugar and other changes i n the tissues rather than with the formation of i c e . By growing Coleus, Saintpaulia and P a l i s o t a f o r a long period at 12° C, they could be made more hardy and able t o tolerate the effects of low temperatures (Spranger, 1941). In passing, i t i s i n t e r e s t i n g to note that some of the deleterious effects of low temperature on growth can be overcome by chemical treatments (Ketellaper, 1963). The nature of the ef f e c t i v e metabolites depends on the species and on the temperature.. For example, n i c o t i n i c acid stimulated the growth of tomato plants at 20/14° C. Cosmos was stimulated by a mixture of vitamin B at 17/10° C and eggplant by a mixture of ribosides at 20/14° C. These active substances did not promote growth at the optimal temperature (Ketellaper, 1963). Many investigations have been made on the temperature responses of grasses. A l l festucoids so f a r examined grow r e l a t i v e l y w e l l at.low temper-atures, have an optimum temperature f o r growth below 27° C, and grow poorly at- 35° C. The panicoid-chloridoid-eragrostoid grasses, on the other hand, have a higher optimum temperature f o r growth, growing vigorously at 35° C and extremely slowly at temperatures below 15° C (Cooper and Tainton, 1968; Evans et a l . , 1964). These differences were consistent when species f o r the two groups were grown under conditions of f l u c t u a t i n g night and day temperatures. Reductions i n growth with lower night temperatures have been observed i n many non-festucoid grasses but i n festucoid species low night temperatures often increase growth (Evans et a l . , 1964). These differences appear to-be connected with the evolutionary o r i g i n and the region of c l i m a t i c adaptation of the two groups. Panicoid and chloridoid-eragrostoid grasses are largely t r o p i c a l i n d i s t r i b u t i o n , while festucoids are primar i l y temperate (Hartley, 1950; Stebbins, 1956; Brown, 1958). Recent comparative biochemical studies have distinguished basic differences between t r o p i c a l and temperate grasses i n the type of carbon path-way i n photosynthesis, the occurrence of photorespiration and the maximum rate of photosynthesis. The panicoid (excluding the temperate, subgenus Dichanthelium, (Downton, et a l . , 1969) ) and the chloridoid-eragrostoid grasses exhibit no photorespiration and have a high maximum rate of photosynthesis. They appear t o f i x most of t h e i r carbon through the C^-dicarboxylic acid pathway (Hatch and Slack, 1966, 1967). They also possess a highly developed parenchymatous sheath around the vascular system i n the leaves (Downton and Tregunna, 1968; Johnson and Hatch, 1968). These bundle sheath c e l l s have r e l a t i v e l y large specialized p l a s t i d s which have few. grana and tend t o accumulate starch (Brown, 1958). Tneir mesophyll c e l l s , on the other hand, contain chloroplasts which are s i m i l a r i n size and structure t o those of plants employing the Calvin cycle only, but these chloroplasts do not accumulate starch. The festucoid species, on the other hand, exhibit photorespiration, lack the C^ pathway, and have a lower maximum rate of photosynthesis. They also lack a specialized parenchymatous sheath around the vascular system. Similar differences i n photosynthetic c h a r a c t e r i s t i c s occur i n several families i n the Dicotyledoneae. Some members of the Amaranthaceae, Portulacaceae, Chenopodiaceae and cer t a i n other Centrospermae exhibit high photosynthetic r a t e s , f i x most of t h e i r carbon through the C^  dicarboxylic acid pathway, and lack photorespiration. They also possess a w e l l developed parenchyma bundle sheath as w e l l as a d e f i n i t e mesophyll layer arranged r a d i a l l y around the sheath. The p l a s t i d s i n the bundle sheath c e l l s are specialized i n accumulation of starch, and l i t t l e or no starch i s accumulated i n the mesophyll c e l l s (Downton and Tregunna, 1968; Tregunna and Downton, 1967). Changes i n starch and pigment content have been observed a f t e r exposure of certain plants t o low temperatures, but these changes have not been extensively studied. To some degree, the effects so f a r observed appear to depend upon the species used and i t s developmental stage. Many investigations have been carr i e d out on the eff e c t s of low temperature on the accumulation of starch and other carbohydrates. D i g i t a r i a decumbens, a t r o p i c a l grass, shows high starch content at 10° C and growth i s severely reduced by t h i s temperature ( H i l l i a r d and West, 1969). I t was suggested that growth reduction was due to i n h i b i t i o n of night translocation. Similar effects were reported f o r Cynodon dactylon (Schmidt and Blaser, 1969; McKell et al.,'1969), Pea pretensis (McKell et a l . , 1969) and Dactylis glomerata Eagles, 1967 and 1967 b; Colby and Drake, 1966; Blaser et a l . , 1966). The accumulation of fructosans at low temperature has been reported f o r several species including Agrostis tenuis (Schmidt and Blaser, 1969) and Lolium perenne (Sullivan and Sprague, 1949). In Poa pratensis and P. compressa, t o t a l carbohydrate accumulation increases under -cold conditions (Brown, 1939). As previously mentioned, the i n h i b i t i o n of the translocation of carbohydrates by low temperatures has been used to explain the accumulation of carbohydrates under these temperatures ( H i l l i a r d and West, 1969). There are several reports which a t t r i b u t e a Qio of one or less t o translocation, and which indicate that the maximum translocation rates occur at low temperature (Went, 1946; Went and Engelberg, 1946; Went and H u l l , 1949; H u l l , 1951; Goodal, 1946). However, most of the accumulated experimental evidence on t h i s point indicates that translocation of carbohydrates has a Q j 0 greater than one, and that i t i s greatest at temperatures about 25° C (Hewitt and C u r t i s , 1948; Swanson and Bohning, 1951; C u r t i s , 1929; Curtis and Herty, 1936; Veron and Aranoff, 1952). Carbohydrates i n plant tissues are involved i n a dynamic system of energy balance. When demand f o r carbohydrates f o r growth exceeds photo-synthesis, there i s an energy l o s s ; conversely carbohydrates accumulate i n tissues when demand i s low compared with photosynthetic carbohydrate production. Thus i t may be postulated that any factor which r e s t r i c t s growth r e l a t i v e l y more than photosynthesis, would cause a carbohydrate accumulation i n the plants. As the r e l a t i v e rate of photosynthesis i s greatest at about 25° C i n temperate species and about 35° C i n t r o p i c a l species, Evans (1964) has suggested that accumulation of assimilates i s u n l i k e l y to occur i n t r o p i c a l species maintained at low temperatures. When stems are c h i l l e d , translocation can be i n h i b i t e d but only during a period of i l l u m i n a t i o n ( H u l l , 1952). Experiments cn the ef f e c t s of pe t i o l e c h i l l i n g on translocation demonstrated l i t t l e or no long-term effects,of cold treatment (Swanson and Geiger, 1967; Geiger, 1969). Many t r o p i c a l plants show severe symptoms of chlorosis and even death when germinated and grown at temperatures between 10° C and 15° C. In temperate plants, on the other hand, chlorophyll remains constant even at the freezing point (Cooper and Tainton, 1968). 8 In Zea, low night temperatures have been found to have a n e g l i g i b l e e f f e c t on chlorophyll concentration (Went, 1957; Alberda, 1969).' However, when the day temperature was reduced to 10° C, concentrations of chlorophyll dropped considerably i n Zea, Pennisetum and Sorghum. In other studies with Zea, the concentration of chlorophyll was n e g l i g i b l e at about 15° C (Friend, 1960 and 1966). An inbred mutant of Zea has been i s o l a t e d which does not form chlorophyll at temperatures as high as 17° C ( M i l l e r d and McWilliam, 1968) . The reduction of chlorophyll concentration i n leaves of Zea was observed m those leaves which elongated during cold treatment (Alberda, 1969) . The primary s i t e of the effects of low temperatures appeared t o be located near the apex of the shoot ( M i l l e r d and McWilliam, 1968). Studies of the e f f e c t s of temperature on p l a s t i d morphology i n Zea indicated abnormal grana development at low temperatures. A s i m i l a r e f f e c t was observed i n the l i g h t at 3° C and at 15° C i n darkness ( K l e i n , 1960; McWilliam and Nay l o r 1967; M i l l e r d et a l . , 1969). The chlorosis of thermophilic plants subjected to low temperature may occur because of a depression i n the synthesis of chlorophyll or i t s immediate precursors, or i t may be caused by t h e i r destruction once they are formed. In Zea, low temperature has been shown to cause a reduction i n the rate of photochlorophyllide synthesis and hence i n the rate of chlorophyllide accumulation (McWilliam and Naylor, 1967). I t i s also known that the enzyme that catalyzes the e s t e r i f i c a t i o n of chlorophyllide to chlorophyll i s sensitive to temperature as w e l l as l i g h t (Wolff and P r i c e , 1957). However, other studies have shown that i n l i g h t at 3° C, Zea transforms protochlorophyll i n t o chlorophyll ( K l e i n , 1960). The i n s i t u absorption rnaximum for chlorophyll at 3° C was shown to d i f f e r from the values obtained from plants grown at 26 C, i n d i c a t i n g a d i f f e r e n t behavior of the chlorophyll produced at low temperature. According to M i l l e r d , the lack of chlorophyll i n a mutant of Zea at 16° C was a consequence of a l e s i o n a f f e c t i n g a r e l a t i v e l y early stage.of either chloroplast development or function ( M i l l e r d and McWilliam, 1968). On t h i s basis, the change i n the absorption maximum of chlorophyll under cold could be explained by the lack of ordered association of the chlorophyll with other components of the photosynthetic apparatus i n the lamellae. Another p o s s i b i l i t y i s that photooxidation of chlorophyll (or i t s immediate precursors such as protochlorophyllide) may occur. Evidence f o r rapid photodestruction has been found i n barley (Augustinussen and Madsen, 1965; V i r g i n , 1955), wheat ( V i r g i n , 1956, 1958) and i n corn (McWilliam and Naylor, 1967; KLein, 1960). In corn f o r example, rapid photooxidation of chlorophyll and carotenoids occurred at 16° C, but the combination of l i g h t and low temperature did not damage the early steps leading to chlorophyll production. Bleaching appeared to r e s u l t not only from the photooxidation of ch l o r o p h y l l , but of chlorophyllide breakdown at a rate greater than t h e i r formation (McWilliam and Naylor, 1967). Once chlorophyll i s formed and presumably complexed within the chloroplast lamellae, i t appears to be protected from photodestruction. A reduction i n carotenoid content may also be a contributing factor i n chlorosis because of i t s r o l e i n the protection of chlorophyll from photooxidation (Anderson and Robertson 1960; G r i f f i t h s et a l . , 1955). 10 2 . The Effects of Temperature and Age on Starch and Pigment .Accumulation i n Triticum, Zea, Phaseolus and Gomphrena . INTRODUCTION The aim of t h i s research was to survey some of the effects of low, non-freezing temperature on starch and pigment concentrations i n plant leaves. I t was hoped that the r e s u l t s of t h i s survey would give some in d i c a t i o n whether plant response to low temperature i s correlated with plant d i s t r i b u t i o n , photosynthetic metabolism and l e a f anatomy, or plant age. To carry out t h i s survey, four species were selected. Two of the species belong to the Monocotyledoneae:Triticum vulgare which carrys out photosynthesis by the conventional Calvin cycle, and Zea mays which possesses the C^-dicarboxylic acid pathway (Downton and Tregunna, 1968). S i m i l a r l y , two species were selected from the Dicotyledoneae: Phaseolus vulgaris and Gomphrena globosa whose pathways of photosynthetic metabolism resemble those °^ Triticum and Zea respectively. Throughout the remainder of t h i s t h e s i s , the Calvin cycle species w i l l be referred to as "temperate", while the C^ species w i l l be c a l l e d " t r o p i c a l " . This terminology i s used f o r convenience since the centers of d i s t r i b u t i o n f o r Calvin cycle and C^ species are located i n the temperate and t r o p i c a l regions respectively (Cooper and Taiton, 1968). I t i s nevertheless recognized that the co r r e l a t i o n between photosynthetic pathway and the d i s t r i b u t i o n of any p a r t i c u l a r species i s not absolute. 11 MATERIALS AND METHODS Seeds of Zea mays L. var. Pioneer, Gomphrena globosa L. var. Amaranth, Triticum vulgare L. var. Spring 'Thatcher and Phaseolus vulgaris var. • Tender Crop Green Pod were obtained from Buckerfield's Seed Co., Vancouver, and were planted i n pots i n a mixture of two-thirds standard greenhouse s o i l and one-t h i r d sand. Three kinds of growth chambers were used f o r t h i s research. One Sherer - G i l l e t t C e l l 255-G Chamber and one Controlled Environments Ltd. EF7 Chamber were used f o r growing most of the plants u n t i l the experiment was started. The Sherer-Gillett Chamber contained eight 80 watt cool white fluorescent lamps and four 25 watt incandescent lamps. I t had an i n t e r i o r 2 f l o o r space of 0.25 m and an i n t e r i o r height of 0.65 m and had no humidity control. The Controlled Environments chamber contained eight 80 watt cool white fluorescent lamps and four 25 watt incandescent lamps. I t had an 2 i n t e r i o r f l o o r surface of 0.65 m and an i n t e r i o r height of 0.97 m. Some plants were also grown i n the Pe r c i v a l chambers described below. For the experimental treatments, the plants were transferred to two . Pe r c i v a l Refrigeration and Manufacturing Co. PGC-78 plant growth chambers. The l i g h t system i n these chambers was composed of sixteen 150 watt cool white fluorescent lamps and ten 25 watt incandescent lamps. A translucent l i g h t b a r r i e r was situated just below the l i g h t system. There was no humidity c o n t r o l , and, accoixiing t o the manufacturer's s p e c i f i c a t i o n s , the humidity varied from 50 to 70% R.H. depending on the ambient condition outside the chamber. The l i g h t i n t e n s i t y during plant growth and during the experimental treatments was approximately 11000 lux at 30 cm above the top of the pots. 12 Measurements showing the v e r t i c a l gradient i n l i g h t i n t e n s i t y i n the Pe r c i v a l growth chambers are given i n Appendix I. The photoperiod was 16 hours throughout t h i s experiment. During growth i n the Shere r - G i l l e t t and Controlled Environment chambers, the day/night temperature conditions were 25-28°/18-22° C as indicated by thermometers suspended i n the chambers. During the experimental treatments i n the P e r c i v a l chamber, 25-28°/18-22° C was used f o r the day/night regime f o r the warm temperature (control) conditions, and 7-ll°/3-8° C were the cold temperature day/night conditions. The plants were watered d a i l y , and NPK nutrient solution (20,20,20 per cent) was applied once a week. For t h i s experiment the plants were transferred t o the warm or cold temperature conditions when they were 10, 21 .or 35 days o l d (15, 26 or 40 days o l d f o r Gomphrena), and they were kept under treatment f o r ten days thereafter. Leaf samples were collected and measurements of pigment and starch content were carried out just before the plants were exposed to the treatment con-d i t i o n s , and at 5 and 10 days a f t e r the treatment was i n i t i a t e d . The design of t h i s experiment was a two factor (temperature and age) f a c t o r i a l , with s p l i t p l o t f o r the three times.at which chlorophyll and starch measurements were made. The layout was a randomized complete block design. Each experimental unit had a r e p l i c a t e number of s i x . Results from preliminary t r i a l s with 3 week old Zea and Triticum plants, which had been exposed to cold or warm temperature treatments f o r one week, were used to determine the number of re p l i c a t e s i n t h i s experiment. For starch determination, a modification of Richer's method was used, i n which starch i s s o l u b i l i z e d with acid and stained with iodine. (Pucher et a l . , 1948; Carter and Neubert 1954; Hassid and Neufeld, 1964). Since 13 starch content in plants varies during the day, preliminary tests were carried out to determine the best time for sample collection. Three tests a day were performed for each species when they were 10 and 21 days old, and grown under the warm condition. For these preliminary tests, starch was estimated out i n the following way. Leaf samples were plunged into boiling water for 30 seconds and then placed i n cold methanol to decolorize. After decolorization, the leaves were floated on an iodine solution prepared as follows: 11 gm of iodine and 22 gm of potassium iodide were dissolved in water and the solution was diluted to 500 ml. 2 ml of this stock solution and 20 grn of potassium iodide were then dissolved i n water and made up to 500 ml. This dilute iodine solution constituted the test solution used (Sandsted et a l . , 1939). Triticum showed no starch i n any of the tests. Zea.showed a slight coloration i n the oldest plants at any time i n the day. Gomphrena and Phaseolus gave positive results at any age and at any time of the day. From t h i s , i t was decided to collect the samples for the main experiment at noon (six hours after the onset of daily illumination). For the starch measurements, a different number of plants was used for each sample according to the species and developmental stages, .as shown below: TABLE 2-1 NUMBER OF PLANTS USED Species 10 Days 21 Days 35 Days Old Old Old Triticum 10 4 1 Zea . 4 1 1 Phaseolus 3 1 1 Gomphrena"* 20 10 1 *For this species the plants were 15, 26 and 40 days old. 14 A f t e r c o l l e c t i o n , the l e a f samples were dried overnight i n an oven at 85-90° C. The following morning they were ground i n a mortar and pestle and weighed. 200 mg dry weight of plant material were used f o r each starch determination. Each weighed sample was then placed i n a 50 ml centrifuge tube and mixed with 4 ml of d i s t i l l e d water. Following t h i s , the tubes were placed i n a b o i l i n g water bath f o r 15 minutes to allow the g e l a t i n i z a t i o n of the starch. They were then cooled i n an i c e bath f o r approximately ten minutes. Three ml of 72% perchloric acid were added and mixed with the plant materia], to s o l u b i l i z e the starch. The mixture was allowed to extract f o r 20 minutes and was then centrifuged. The supernatant was poured i n t o a 50 ml volumetric f l a s k , and 3 ml of acid were added and mixed with the residue to complete the extraction. A f t e r 20 minutes, the samples were centrifuged again and the supernatants were combined and d i l u t e d to 50 ml with d i s t i l l e d water. Supernatants stored at 4° C showed no change i n absorbance f o r two days a f t e r extraction. Extracts were further d i l u t e d when the soluble starch concentration was very high. 0.05 ml'of iodine solution prepared from 15 ml of the stock solution described previously and 8 gm of potassium iodide dissolved i n 200 ml d i s t i l l e d water, were added to 5 ml of the extract, and the absorbance of t h i s mixture was immediately measured i n a Beckman DU spectrophotometer at 620 nm. The amount of starch present was determined by r e f e r r i n g to a standard curve, which was prepared using p u r i f i e d potato starch (ACS) treated according to the above methods (Figure 2-1). The procedure f o r the extraction and measurement of pigments was based on the method of Bruinsma (1963). Leaf samples were collected and t h e i r surface area and fresh weight was determined. The s i z e of the sample used depended on plant age and species as follows: TABLE 2-2 FRESH WEIGHT (gm) OF LEAF SAMPLES FOR PIGMENT DETERMINATION Plant Age - Days  Species 10 21 3_5 I r i t i c u m 0.3 (32.5)* 0.2 (32.5) 0.1 (50.0) Zea 0.5 (32.5) 0.3 (53.5) 0.5 (95.0) Phaseolus 0.5 (17.5) 0.3 (31.0) 0.5 (32.5) Gomphrena5-" 0.2 ( 2.5) 0.3 ( 3.76) 0.2 (10.00) ""Numbers i n parenthesis are the heights of the plants i n cm. **In t h i s species, the plants were 15, 26 and 40 days old. Figure 2-1 Effect of potato starch concentration on the absorbance of aqueous iodine-potassium iodide-starch mixture at 620 nm. S o l i d and open, c i r c l e s represent r e p l i c a t e determinations ; at.each"concentration tested. 16 Q.O i ' I I 1 M I III . I 0 5 10 15 20 25 30 STARCH CONCENTRATION (mg/50 ml solution) 17 For the ten day old plants, entire leaves were used, but f o r the older plants, the le a f samples consisted of segments cut from the central portion of the l e a f (intermediate between the l e a f base and t i p ) . " Each l e a f sample was cut in t o small pieces and mixed with 50 ml of cold (4° C) 80% aqueous acetone (reagent grade ACS), the mixture'was then blended at high speed i n an Osterizer blendor f o r 0.5 to 2 minutes and the re s u l t i n g s l u r r y was quantitatively f i l t e r e d i n t o a volumetric f l a s k . The residue on the f i l t e r was washed several times with additional cold 80% aqueous acetone u n t i l a l l the chlorophyll was extracted. The extract was di l u t e d to 100 ml (200 ml for Zea samples) by adding more cold 80% aqueous acetone. The extracts were then stored i n darkness at 4° C.** u n t i l spectro-photometric analysis was carried out. To determine the concentration of chlorophyll a, chlorophyll b and t o t a l chlorophyll, 5 ml of the aqueous acetone extract was used and i t s absor-bance was measured at 645, 652 and 663 nm using a Beckman DU spectrophotometer. The pigment concentrations were then calculated using the following equations (Bruinsma, 1963): 1) Chi. a (mg/1) = 12.7 ODg 6 3 - 2.7 OD^ 2) Chi. b (mg/1) = 22.9 O D ^ - 4.7 ODg 6 3 W + 8'0 2 H>63 3) Total Chi. (mg/1) = 20.20 0D e i l C + 8.02 0DC 4) Total Chi. (mg/1) = 1000 Q D642 36.0 Where OD ( o p t i c a l density or absorbance) = log I and I Q are the i n t e n s i t y of the transmitted and incident l i g h t respectively. Formula (3) resulted from the addition of 1 and 2. The absorption curves f o r chlorophyll a and chlorophyll b ( s p e c i f i c absorption c o e f f i c i e n t vs. wavelength) intersect at * See App. I I ** See App. I l l 18 652 nm at a common s p e c i f i c absorption c o e f f i c i e n t a of 36.0. The amount of t o t a l chlorophyll can therefore also be determined by measuring the absorbance at 652 nm and computing the concentration of t o t a l chlorophyll as given i n Formula 4. The average of the r e s u l t s obtained using Formulae 3 and 4 were used to compute the t o t a l chlorophyll concentration. The solution used f o r chlorophyll determination was also u t i l i z e d f o r the t o t a l carotenoid estimation. Solution absorbance was measured i n the spectrophotometer at 440.5 nm and the following equation was used (D. von Wettstein, 1957): Car. (mg/1) = 4.695 O P ^ ^ - 2.68 C ( a + fc) Leaf pigment concentrations were expressed both on a fresh weight and l e a f area basis. In t h i s way, the more constant unit i n the cold and warm conditions could be used t o compare the data. A m u l t i f a c t o r i a l analysis of variance was carried out to determine the s i g n i f i c a n t effects of age, temperature and duration of treatment, whenever differences were detected at the 5% l e v e l , Duncan's new multiple range t e s t was carr i e d out to determine which conditions d i f f e r e d from each other. For each set of r e p l i c a t e s , 95% confidence l i m i t s were also calculated, and these are indicated by the v e r t i c a l l i n e s about sample means i n many of the figures i n the following sections. 19 RESULTS (i) Effects of temperature and age on starch content. Figures 2-2 and 2-3 show that in warm conditions, the accumulation of starch was consistently low except in Phaseolus throughout a l l the ages tested, and in Zea in the 20-21 day old plants. In Phaseolus, the mean starch concentrations ranged from 563 to 982 mg per g dry weight and were high in both the primary and trifoliate leaves. In Zea, a peak of over 300 mg starch per gm dry weight was observed at about 20 days, and younger and older plants contained significantly less starch. In a l l other cases, the mean starch concentrations were-below 200 mg/g in the warm temperature conditions. In Triticum starch was practically absent; the highest concen-tration observed was about 30 mg/g in the youngest plants. In Gomphrena, the highest concentration found was 153 mg/g in the 26 day old plants, and the younger plants contained significantly less starch. Low temperature tended to increase starch accumulation and the magnitude of the increase depended on plant species, age, and time of exposure to cold. Gomphrena showed a very great increase in starch concentration in response to cold, and the quantity of starch accumulated increased significantly when the time of exposure to cold was extended from 5 to 10 days. In Phaseolus, small significant differences in starch concentrations occurred in the youngest plants after 5 days of treatment, and in the inter-mediate age. In this case, there was no significant effect of duration of cold treatment. Triticum exhibited significantly higher starch accumulation in the cold except in the youngest plants used after 5 days of treatment. In the intermediate age, the concentration of starch accumulated increased with time of exposure to cold, but even under cold conditions, only quite low levels of starch content were attained. In Zea, cold had significant Figure 2-2 Starch concentrations i n 10, 21 and 35 day-old Triticum (a) and Zea (b) plants subsequently exposed to warm and cold conditions f o r 0, 5 or 10 days. © Warm temperature O Cold temperature T I M E ( d a y s ) Figure 2-3 Starch concentrations i n 10, 21 and 35 day-old Phaseolus (a) plant and 15 and 26 day-old Gomphrena (b) plants subsequently exposed to warm or cold conditions "for 0, 5 or 10 days. e Warm temperature ' o Cold temperature 21 1200 1000 800 5 600 400 - 1200 S IOOO 800-600 400 200 40 45 T I M E ( d a y s ) 22 e f f e c t s i n the two youngest ages, but not i n the oldest plants. In the youngest age, the differences between the warm and cold treatment were not great, but i n the intermediate age, the starch concentration i n the cold plants remained constant while i n the warm plants i t dropped appreciably, thus r e s u l t i n g i n large differences. ( i i ) The Effects of Temperature and Age on Pigment Accumulation, a. Triticum vulgare The behavior of Triticum depended to some extent on the units used to express chlorophyll concentration. As shown i n Figures 2-4 and 2-5, the t o t a l chlorophyll arid chlorophyll a and chlorophyll b concentrations per gram fresh weight increased greatly i n the 26 to 31 day o l d plants. Following t h i s increase, high levels of chlorophylls were maintained i n the older plants. Although the chlorophyll concentrations per gram fresh weight were s l i g h t l y lower f o r the cold-treated plants, the eff e c t of temperature was not s i g n i f i c a n t at the 5% l e v e l . When the chlorophyll concentrations were expressed on a l e a f area basis, however, no increase i n the l e v e l of chlorophylls was evident u n t i l the plants were 40 to 45 days old. Once again, low temperature did not s i g n i f i c a n t l y a f f e c t the t o t a l chlorophyll or chlorophyll a and b concentrations. The t o t a l carotenoid l e v e l was not s i g n i f i c a n t l y affected by temperature. Carotenoid content increased with age when expressed on a per gram fresh weight ba s i s , but did not change appreciably when l e a f area was the basis of expression. Figure 2-4 Total chlorophyll (a) and to t a l carotenoid (b) concentrations i n 10, and 35 day-old Triticum plants subsequently exposed to warm or cold conditions for 0, 5 or 10 days. ©A Warm temperature OA Cold temperature Figure 2-5 Chlorophyll a (a) and chlorophyll b (b) concentrations i n 10, 21 and 35 day-old Triticum plants subsequently exposed to warm or cold conditions for 0, 5 or 10 days. ©A Warm temperature OA Cold temperature 25 b. Zea mays Figures 2-6 and 2-7 show the chlorophyll and carotenoid concentrations i n leaves of Zea after warm or cold temperature treatments at different ages. At the warm temperature., the to t a l chlorophyll concentration generally tended to decrease as the plants aged, with a particularly large drop occurring i n plants which were older than 31 days. On exposure to cold temperature, the total chlorophyll concentration was reduced i n a l l the ages studied. Quite similar responses were found i n the two youngest ages studied. For these plants, the difference i n chlorophyll concentration between the warm and cold temperature treatments increased with the duration of the treatment and were significant at the 5% level. For the oldest set of plants, however, cold treatment had. l i t t l e or no significant effect on t o t a l chlorophyll accumulation. The small difference which was evident after 10 days of treatment of the oldest plants was not significant i f the chlorophyll concentrations were expressed on a leaf area basis. Figure 2-7 separates the effects of temperature and age on chlorophylls a and b. The changes i n chlorophyll a closely followed the changes i n tot a l chlorophylls. This was expected since chlorophyll a constituted 60-90% of the to t a l . Chlorophyll b, on the other hand, was less sensitive to temperature than chlorophyll a, although i t also exhibited a decline with age. For chlorophyll b, low temperature caused a significant reduction only i n the two youngest sets of plants, and then only after they had been under treatment for 10 days. Figure 2-6 shows that carotenoid concentration remained relatively con-stant and was not significantly affected by temperature or age. Figure 2-6 Total chlorophyll (a) and to t a l carotenoid .(b) concentrations i n 10, 21 and 35 day-old Zea plants subsequently exposed to warm or cold conditions for 0, 5 or 10 days. ©A Warm temperature OA Cold temperature 1 Figure 2-7 Chlorophyll a (a) and chlorophyll b (b) concentrations i n 10, 21 and 35 day-old Zea plants subsequently exposed to warm or cold conditions for 0, 5 or 10 days. O A Warm temperature OA Cold temperature 28 c. Phaseolus vulgaris -Figure 2-8 indicates that the t o t a l chlorophyll concentration i n the warm Phaseolus plants did not change greatly with age. The t o t a l chlorophyll level was lower i n the cold-treated plants i n a l l cases, but this reduction was significant only for the two oldest sets of plants after both 5 and 10 days of exposure to cold. The size of the reduction i n chlorophyll con-centration was similar for cold treatments of 5 or 10 days duration. Figure 2-9 shows that the reduction i n tot a l chlorophyll i n Phaseolus may be attributed to reductions i n both chlorophyll a and chlorophyll b. The to t a l carotenoid concentration was not significantly affected by age or cold treatment. . d. Gomphrena globosa Figure 2-10 shows that when the results were expressed on a per gram fresh weight basis, there was a general increase i n total chlorophyll con-centration during development of Gomphrena i n warm conditions. Cold treatment reduced the total level of chlorophyll at a l l ages, and the reduction was greatest i n those plants which were exposed to cold for 10 days. The difference i n t o t a l chlorophyll concentration between the warm and cold treated plants were significant except for .the two oldest sets of plants when they had been under cold treatment for only 5 days. This-response of tot a l chlorophyll to cold temperature was related to reductions i n the levels of both chlorophyll a and chlorophyll b as indicated by the results i n Figure 2-11. When the total chlorophyll, chlorophyll a and chlorophyll b concentrations were expressed per unit leaf area, a very different pattern was obtained. Total chlorophyll content remained relatively constant except i n the oldest plants Figure 2-8 Total chlorophyll (a) and to t a l carotenoid (b) concentrations i n 10, and 35 day-old Phaseolus plants subsequently exposed to warm or cold conditions for 0, 5 or 10 days. ©A Warm temperature O A Cold temperature Figure 2-9 Chlorophyll a (a) and chlorophyll b (b) concentrations i n 10, 21 and 35 day-old Phaseolus plants subsequently exposed to warm or cold conditions for 0, 5 or 10 days. © A Warm temperature O A Cold temperature Figure 2-10 Total chlorophyll (a) and t o t a l carotenoid (b) concentrations i n 15, 26 and 40 day-old Gomphrena plants subsequently exposed to warm or cold conditions for 0, 5 or 10 days. O A Warm temperature OA Cold temperature Figure 2-11 Chlorophyll a (a) and chlorophyll b (b) concentrations i n 15, 26 and 40 day-old Gomphrena plants subsequently exposed to warm or cold conditions for 0, 5 or 10 days. ©A Warm temperature OA Cold temperature 33 which contained s l i g h t l y higher l e v e l s . No s i g n i f i c a n t response to cold was evident i n any case. Whatever the mode of expression, the t o t a l carotenoid concentration did not change s i g n i f i c a n t l y i n response to increasing age or cold temperature. DISCUSSION In summary, low, non-freezing temperatures caused s i g n i f i c a n t effects on starch and chlorophyll accumulation, but not on carotenoid l e v e l s . The in t e n s i t y of the effects varied according to species, developmental stage, and time of exposure to cold. A l l of the species examined showed s i g n i f i c a n t l y more starch i n the cold condition on at least one occasion during t h i s experiment. Starch i s generally known t o accumulate to higher levels i n the Dicotyledoneae than i n Monocotyledoneae (Smith, 1969; Gates and Simpson 1966) and the present resu l t s are i n agreement with t h i s r e l a t i o n s h i p . An explanation f o r the absence, or presence only i n trace amounts, of starch i n the Monocotyledoneae may l i e i n the presence of large amounts of fructose and polyfructosans, which also accumulate i n greater amounts under cold temperature (Schmidt and Blaser, 1969). I t i s also known that within the grasses, t r o p i c a l species tend to accumulate starch, while the temperate ones largely accumulate fructosans. The present r e s u l t s correlate w e l l with these findings since Zea accumulated s i g n i f i c a n t l y higher levels of starch than d i d Triticum. Because the optimum temperature f o r photosynthesis i s lower f o r temperate species than f o r t r o p i c a l ones, Evans (1964) has suggested that i n most cases photosynthesis would be more reduced by cold conditions i n t r o p i c a l ,v?'-' • 34 species than i n temperate plants. As a result, starch accumulation would be unlikely to occur i n tropical species at low temperature. However, Evans also pointed out that the growth of tropical species may be greatly reduced by cold conditions, while cold temperatures, particularly at night, sometimes increase the growth of temperate species. From the effects of cold temperature on growth alone, i t would be plausible to suggest that starch accumulation would occur i n those plants whose growth i s more restricted by cold temperature, . namely the tropical species. Therefore, both the photosynthetic production of carbohydrates and their u t i l i z a t i o n i n growth must be taken into account. It would be expected that starch w i l l tend to accumulate, whenever growth i s reduced by cold to a greater extent than photosynthesis. Since a l l 'of the species tested exhibited higher levels of starch i n cold conditions than i n warm, the results obtained do not permit a ready distinction between temperate and tropical species on the basis of starch accumulation at low temperature. In the two Monocotyledoneae studied, Zea appeared to be more sensitive to cold at the youngest age tested than was Triticum, while i n the oldest plants tested the reverse occurred. In any case, when cold treatment resulted i n significant differences i n starch concentration, the magnitudes of the differences (g starch/g dry weight) were much larger i n Zea than i n Triticum. Cold temperature caused a greater reduction i n height of Zea than Triticum, and this may partly explain the greater accumulations of starch i n Zea. On the other hand, the per cent increase i n starch concentration at low temperature was smaller i n Zea than i n Triticum. This may be accounted for by the very low starch concentration i n Triticum at the warm temperature. 35 The Dicotyledoneae studied also d i f f e r e d with respect to the e f f e c t of temperature on starch accumulation. Gomphrena showed extremely large increases i n starch concentration i n cold conditions i n the two ages tested, while Phaseolus exhibited r e l a t i v e l y small s i g n i f i c a n t increases i n the young and intermediate ages only. I t should be noted that since Gomphrena has a r e l a t i v e l y long l i f e c y cle, only the early stages i n i t s development were exajnined i n t h i s experiment. As i n the case of the Monocotyledoneae, the t r o p i c a l species (Gomphrena) showed a larger percentage reduction i n growth i n the cold condition (26.29%) than d i d the temperate Phaseolus (12.91%). Figure 2-3 indicates that some of the starch concentrations observed with Gomphrena and Phaseolus exceeded 1 g per g dry weight. Two reasons can be advanced to explain t h i s unusual r e s u l t . F i r s t of a l l , the iodine-starch complex f o r Gomphrena and Phaseolus may be more ef f e c t i v e i n absorbing 620 nm l i g h t than was the potato starch-iodine standard. To avoid t h i s , the method of starch measurement could be improved by extracting starch from each species and using these extracts to prepare a standard starch curve f o r i ^ each species. This change i n method, however, would s t i l l not avoid the p o s s i b i l i t y that the structure of starch and i t s a b i l i t y to complex with iodine may be affected by the temperature of starch synthesis. Secondly, the high starch concentrations i n Gomphrena and Phaseolus made i t necessary to d i l u t e the plant extracts before spectrophotometric readings could be taken. I t i s possible that these d i l u t i o n s may have introduced some error i n t o the starch determinations. The r e s u l t s show c l e a r l y that the e f f e c t of low, non-freezing temperature on chlorophyll concentration was also dependent on species, developmental . 3 6 stage and duration of treatment. In some instances, cold temperature had no s i g n i f i c a n t e f f e c t on pigment l e v e l s , but whenever a s i g n i f i c a n t response was observed, i t involved a decrease i n the concentration of chlorophylls. In addition, the effects of cold on t o t a l chlorophyll concentration can be attributed to the p a r a l l e l responses of both chlorophyll a and chlorophyll b. In the Monocotyledoneae studied, Zea exhibited a s i g n i f i c a n t reduction i n chlorophyll concentration under cold conditions. This r e s u l t i s i n agreement with the findings of several other studies (Alberda, 1969; Friend, 1966; M i l l e r d and McWilliam, 1968; McWilliam and Naylor, 1967) but most of these previous studies used only very young seedlings. Triticum, on the other hand, exhibited no s i g n i f i c a n t response i n chlorophyll concentration to cold temperature treatment. This too i s i n agreement with previous indications (Friend, 1961; Friend, 1966). These differences i n pigment response to low temperature were correlated with differences i n growth. Cold temperature treatment caused a much larger reduction i n growth and increase i n starch concentration i n Zea than i n Triticum, and i t seems reasonable to suggest that lower growth rate of Zea r e f l e c t e d lower CO2 f i x a t i o n caused by pigment l i m i t a t i o n s to photosynthesis. This may not be the only reason, however. Short-term gas exchange studies have shown that the optimum temperature f o r photosynthesis i n Zea i s about 39° C while f o r Triticum i t i s about 25° C ( J o l l i f f e , 1970). The lower growth rate of Zea may therefore also be due to the fact that at cold temperature i t i s much farther from optimum con-dit i o n s than Triticum. In the Dicotyledoneae used, both Phaseolus and Gomphrena exhibited s i g n i f i c a n t reductions of chlorophyll concentration i n the cold. In Gomphrena, 37 cold treatment caused a greater reduction i n growth but a smaller reduction i n chlorophyll concentration than i n Phaseolus. Thus the effects of cold were quite different from the grasses. It i s possible that this difference i s only apparent, for with Phaseolus measurements were made during much of i t s l i f e cycle, while i n Gomphrena, which has a relatively long l i f e cycle, only the early developmental stages were observed. In the case of Gomphrena the pigment results were different according to the mode of expression used. Chlorophyll accumulation was significantly less i n some of the cold treatments when the results were expressed on a fresh weight basis, but no significant response was evident when leaf area was the basis of expression. In other words, under cold temperature, the ratio of chlorophyll concentration to fresh weight declined while the ratio of chlorophyll concentration to leaf area remained constant. Therefore, differences such as these can be attributed to the effects of age and cold treatment on the ratio of leaf fresh weight to leaf area. In the three species which exhibited significant reductions i n chlorophyll concentration i n the cold, the extent of the reduction varied according to plant age and the duration of treatment. While i n the case of Gomphrena similar reductions i n chlorophyll concentration per g fresh weight were found at a l l ages studied, the greatest reduction i n Zea occurred i n the two youngest ages, and i n Phaseolus the intermediate age, which just preceded flower development, was the most sensitive. In a l l three cases, the reductions i n chlorophyll level coincided with periods of development marked by the 'initiation and expansion of many young leaves. Significant effects of the duration of cold treatment were evident i n only 3 cases. The two youngest sets of Zea, and the intermediate Gomphrena plants showed 38 reductions i n total chlorophyll concentration between 5 and 10 days of treatment which were significant at the 5 per cent level. To some extent, the effect of low, non-freezing temperature on starch concentration appear to be inversely related to i t s effect on chlorophyll concentration. For example, i n Zea the two youngest ages exhibited significant increases i n starch concentration and decreases i n chlorophyll concentration under cold treatment. Similar changes occurred i n Gomphrena. In Phaseolus, however, an increase i n starch concentration and a reduction i n chlorophyll concentration occurred only i n the intermediate age. In Triticum, there was only a small increase i n starch concentration .in the cold condition, while the chlorophyll concentration was not significantly affected. I t must also be pointed out that the interpretation of the changes i n pigment concentration i s d i f f i c u l t i n those cases where large changes i n the starch concentration occurred. Far' example, i n Gomphrena the change i n starch concentration during the cold treatments was of the order of one gram per gram dry weight. That i s , the dry weight of Gomphrena had l i t t l e starch at the start of the cold treatment, but was mostly starch after 10 days.of cold. I t would be expected that this large increase i n starch concentration would be reflected by decreases i n the proportional con-centrations of other components of the dry matter of the plant. The often inverse relationship between the changes i n starch and chlorophyll concentrations i n the species tested was pointed out i n the previous paragraph and may be based on this sort of effect. Examination of the starch results indicates that increases i n starch concentration may have been sufficiently large to contribute to the decreases i n chlorophyll concentration observed iri 'Gomphrena and i n the intermediate ages of Phaseolus and Zea. There does not seem to be any convenient way of directly expressing the starch and chlorophyll results relative to each other. Perhaps, i f these studies are continued, the results could be made relative to protein content, c e l l number, or some other possibly independent index. In these:experiments, carotenoid concentration was unaffected'by. cold or age. This may indicate that low temperatures do not influence the postulated role of carotenoids i n preventing chlorophyll photooxidation. Confirmation of this aspect must await studies of the interactions of individual carotenoid pigments with cold temperature and chlorophyll concentration. In conclusion, depending on species, age, and duration of treatment, low non-freezing temperature caused high starch or low chlorophyll concentrations to occur, or i n some cases the cold had no significant effect. The effects of cold on starch and chlorophylls i n the tropical species used were often greater than i n the temperate species. This was particularly true when the youngest ages only are considered, but this relationship was sometimes reversed i n the oldest plants. Therefore, general correlations between low temperature effects and photosynthetic metabolism or higher systematic categories are not evident from these results. Starch accumulation at low temperature may be the result of an excess of photosynthesis over growth, while low temperature may reduce chlorophyll accumulation by inhibiting i t s synthesis or promoting i t s breakdown. 40 3. The Effects of Temperature and Kinetin on the Accumulation of Chlorophyll i n E t i o l a t e d Zea leaves INTRODUCTION The preceding experiment was l i m i t e d i n scope by the r e l a t i v e l y long time required f o r the starch and pigment analyses. As an alt e r n a t i v e to the extractive determination of pigment concentration, a procedure was developed f o r the rapid i n d i c a t i o n of pigment concentration during the greening of e t i o l a t e d leaves. This procedure was based on the photometric determination of the transmission of 665 nm l i g h t through the leaves. F u l l y green leaves could not be used since l e a f l i g h t transmission i s only s l i g h t l y affected by moderate changes i n pigment concentration i n green leaves. This method was applied to a study of the greening process i n e t i o l a t e d Zea leaves i n both warm and cold temperatures. Zea was chosen since the previous experiment and other studies (Alberda, 1969; Friend 1966; McWilliam and Naylor, 1967; M i l l e r d and McWilliam, 1968) had i d e n t i f i e d the s e n s i t i v i t y of pigment leaves to cold temperature i n young leaves. A series of te s t s was carri e d out to determine the k i n e t i c s of pigment accumulation i n et i o l a t e d leaves during the f i r s t 24 hours of i l l u m i n a t i o n at warm or cold temperatures. To es t a b l i s h the re l a t i o n s h i p between l i g h t transmission and chlorophyll concentration, chlorophyll extractions were carri e d out to complement the transmission measurements. F i n a l l y , the p o s s i b i l i t y of using k i n e t i n to overcome the i n h i b i t o r y e f f e c t of cold temperature on chlorophyll accumulation was tested. MATERIALS AND METHODS Zea mays L. var. Pioneer plants were grown i n the same way as before except that no i l l u m i n a t i o n was supplied so the plants remained e t i o l a t e d . When the f i r s t l e a f was about 4 cm long, the plants were exposed to con-tinuous l i g h t i n the P e r c i v a l growth chambers. Following the s t a r t of il l u m i n a t i o n , the plants were exposed to d i f f e r e n t periods of treatment i n warm (25 - 28° C) or cold (7 - 11° C) temperature conditions. The apparatus f o r measuring the absorption of l i g h t by the greening leaves included a white l i g h t source which provided approximately 1.6 x 4 -2 -1 10 ergs cm sec at the surface of the l e a f . The l i g h t beam was intercepted by a red f i l t e r (Balzers B-40, 665 nm, with a band width of 11 nm) before i t reached the l e a f . A l i g h t detector (PIN photodiode-Hewlett Packard HP 5082-4220) was placed on the other side of the l e a f to detect the transmitted l i g h t . The l i g h t sensor was connected to an amplifier from which the readings were taken. When measuring the l i g h t transmitted through the l e a f , the l e a f which developed f i r s t was cut and placed between two microscope s l i d e s , and these were then placed between, the l i g h t source and the l i g h t sensor i n such a way that the beam was incident at approximately the centre of the l e a f . Ten measurements from ten d i f f e r e n t leaves were made under each set of conditions. The l e a f thickness was measured a f t e r the transmission reading was fini s h e d . For t h i s purpose a micrometer which had a Vernier mechanism allowing measurements to ten thousanths of an inch was used. Chlorophyll determinations were c a r r i e d out as previously described and s i x r e p l i c a t e determinations were made f o r each condition. 42 For the kinetin experiments, 6-furfurylaminopurine (A grade M.W. 2152 from CalBiochem) i n a concentration of 20 mg/1 was sprayed on to the plants at the beginning of the experiment u n t i l the leaves were wet. RESULTS AND DISCUSSION Figure 3-1 shows the time course of leaf light transmission after etiolated Zea leaves were exposed to 11000 lux illumination i n warm and cold temperatures. After a two hour lag period, which was also observed i n subsequent tests, the transmission of light by the warm leaves declined rapidly with time. A logarithmic regression curve with the equation shown on Figure 3-1 was f i t t e d to the data. To account for the two hour lag period, the f i r s t point (time = o) was omitted from the regression. The 2 high degree of correlation between the regression and the data (r =0.94) confirmed the exponential nature of the decrease i n leaf light transmission. I f i t i s assumed that the leaf posesses a constant thickness and that i t behaves as a pure pigment solution, then according to Lambert's law the exponential decrease i n transmission could be the result of a linear increase i n pigment concentration. As w i l l be seen later, a linear increase i n chlorophyll concentration was actually observed during the i n i t i a l 24 hours of greening. For the leaves which were exposed to cold conditions, however, the light transmission values remained constant. The sample variation for the cold treated plants was relatively large. It was observed that on exposure to cold, some of the leaves suffered p a r t i a l bleaching of their i n i t i a l l y pale yellow colour, and this may have resulted i n some high ligh t trans-mission readings. Figure 3-1 Changes i n the per cent transmission of light through leaves during the i n i t i a l 48 hours of greening of etiolated Zea seedlings exposed to warm or cold conditions. B Warm temperature • Cold temperature 44 Since leaf thickness, and therefore leaf light transmission might have been influenced by temperature, the thickness of each leaf was recorded. Figure 3-2 demonstrates that temperature and treatment time had no significant effect on leaf thickness during the period of study. Therefore, the observed variations i n leaf light transmission during this experiment were not the result of changes i n leaf thickness. Figure 3-3 shows the results obtained from chlorophyll extractions carried out at different times during the warm temperature treatment. Only the values for chlorophyll a are shown, because i t i s the f i r s t chlorophyll formed during greening and i t represents up to 90% of total chlorophyll. The•cold treated plants did not accumulate any significant quantity of chlorophyll. In the plants exposed to warm temperature, there was a continuous increase i n chlorophyll a concentration with time, and this increase could be described by the linear regression equations given i n Figure 3-3. A better correlation was obtained i f the chlorophyll a concentration was expressed on a per gram fresh weight basis rather than on a leaf area basis. In addition, Figure 3-4.shows the relationship, between chlorophyll a concentration and leaf light transmission. For both modes of expression of chlorophyll a concentration, a correlation coefficient of 0.99 was obtained, indicating that variations i n leaf li g h t transmission are almost entirely attributable to changes i n chlorophyll a concentration. Therefore, this relationship can be used to predict leaf chlorophyll concentration from measurements of leaf li g h t transmission during greening. Figure 3-5 summarizes the results of a series 'of measurements of li g h t transmission by etiolated Zea leaves which were illundnated and kept Figure 3-2 Variations i n leaf thickness during the i n i t i a l 48 hours of greening of etiolated Zea seedlings exposed to warm or cold conditions. a Warm temperature D Cold temperature 1 Figure 3-3 Changes i n chlorophyll a concentration during, the i n i t i a l 48 hours of greening of etiolated Zea seedlings i n warm conditions. Figure 3-4 Relationship between the per cent transmission of l i g h t through leaves and the leaf chlorophyll a concentration during the greening of etiolated Zea seedlings i n warm conditions. 47 CO % CHLOROPHYLL a CONCENTRATION (|jg/g) co < cn Figure 3-5 Changes i n the per cent transmission of light through leaves during the i n i t i a l 24 hours of greening of etiolated Zea seedlings. c Warm temperature for entire 24 hours O Cold temperature for entire 24 hours A Warm temperature for i n i t i a l 2 hours, remainder cold o Warm temperature for i n i t i a l 4 hours, remainder cold A Cold temperature for i n i t i a l 2 hours, remainder warm • Cold temperature for i n i t i a l 4 hours, remainder warm 80r 101 i . . 1 < < • « • • -0 4 8 12 16 20 24 TIME (hours) for different periods of time i n warm or cold conditions. As before, the plants which were kept i n the cold for the entire period showed no significant change i n leaf light transmission. In the warm condition, there again appeared to be a lag of about two hours before the leaf light transmission values decreased significantly. Plants which were exposed to warm temperature for two hours and then transferred to the cold did not exhibit any significant change i n leaf light transmission. The presence of an i n i t i a l lag before the onset of chlorophyll accumulation i s i n agreement with earl i e r reports (Virgin, 1955; Kirk, et a l 1967). Plants which were exposed to warm temperature for four hours showed dist i n c t l y lower values of leaf light transmission, indicating that the accumulation of chlorophyll had started by that time. However, when plants were then transferred to the cold temperature, leaf lig h t trans-mission quickly Increased u n t i l after eight hours of illumination i t was equivalent to the values exhibited by leaves which had been i n continuous cold treatment. This result indicates that the pigment synthesized during the warm temperature treatment was destroyed, or was synthesized at a slower rate than i t s destruction, i n the cold. Previous studies have also indicated a rapid destruction of chlorophyll under cold temperature i n corn (MacWilliam and Naylor, 1967; KLein, 1960) i n wheat (Virgin, 1956 and 1958) and i n barley (Augustinussen and Madsen, 1965; Virgin, 1955). On the other hand, when the plants were exposed for two or four hours of cold treatment at the beginning of illumination, no significant decline i n leaf lig h t transmission was evident u n t i l they had subsequently been i n warm conditions for eight or ten hours respectively. Therefore the i n i t i a l lag i n chlorophyll accumulation i n warm temperature was extended by 50 preceding cold treatment. Nevertheless, once chlorophyll began to accumulate following the i n i t i a l cold treatment, the kinetics of accumulation resembled those observed with the plants which were exposed to warm temperature throughout. These results may indicate that the i n i t i a l cold treatment may have retarded that step of chloroplast development which i s coincident with the end of the lag phase. The early steps of chloroplast formation are known to occur during the lag phase (Kirk, 1967). At the end of the lag phase, chloroplast structure undergoes specific changes following which chlorophyll accumulation parallels chloroplast development. It i s possible that the i n i t i a l cold treatment may prevent these structural changes i n chloroplasts by causing the accumulation of inhibitory substances or by some other mechanism. Once the structur'al changes have occurred and the lag phase i s ended, chlorophyll accumulation seems to be independent of the i n i t i a l , cold treatment. Figure 3-6 shows that the application of kinetin did not significantly a l t e r leaf lig h t transmission i n either the warm or the cold condition. Thus, kinetin did not overcome the inhibitory effect of cold temperature on greening, even though kinetin i s known to suppress the loss of chlorophyll from dark-ened disks of mature green Xanthium leaves (Osborne and McCalla, 1961). In summary, the technique of measuring leaf light transmission i s a convenient and rapid method of determining chlorophyll concentration i n greening leaves. It i s particularly useful i n studies of the effects of various factors on the kinetics of chlorophyll accumulation, and i n future i t might be adapted to continuous measurements on the same leaf. In the present study, this technique has been used to show that cold temperature extends the lag phase i n chlorophyll accumulation, and causes a net decrease i n chlorophyll concentration which i s not prevented by kinetin treatment. Figure 3-6 Effect of kinetin on the changes i n per cent transmission of light through leaves during the i n i t i a l 24 hours of greening of etiolated Zea leaves exposed to warm or cold conditions. © Warm temperature •a Warm temperature plus kinetin O Cold temperature a Cold temperature plus kinetin P E R C E N T T R A N S M I S S I O N Cn 52 4. Literature Cited 1. Alberda, T.H. 1969. The effect of low temperature on dry matter production, chlorophyll concentration and photosynthesis of maize plants of different ages. Acta Bot. Neerl. 18(1): 39-49. 2. Anderson, I.C. and D.S. Robertson. 1960. The role of carotenoids i n protecting chlorophyll from photodestruction. Plant Physiol. 35: 531-534. 3. Arnon, D.I. 1949. Copper enzymes in isolated chlorophasts. Plant Physiol. 24: 1-15. • 4. Ashby, W.C. and H. Hellmers. 1959. Flowering and growth responses to photoperiod and temperature for six southern Califomian grasses. Bot. Gaz. 120: 151-157. 5. Augustinussen, E. and A. Madson. 1965. 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Plantarum 8: 630-643. 78. Virgin, H.I. 1956. Light-induced stomatal transpiration of etiolated wheat leaves as related to chlorophyll a content. Physiol. Plantarum 9: 482-493. 79. Virgin, H.I. 1958. Studies on the formation of protochlorophyll and chlorophyll a under varying light treatment. Physiol. Plantarum 11: 347-362. 80. Went, W.F. 1944. Plant growth under controlled conditions. II. Thermoperiodicity i n growth and fr u i t i n g of the tomato. Amer. J. Bot. 31: 135-150. 58 •81. Went, F.W-. and R. Engelsberg. 1946. Plant growth under controlled conditions. VII. Sucrose content of the tomato plant. Arch, Biochem. 9: 187-200. 82. Went, F.W. and H.M. Hull. 1949. The effect of temperature upon translocation of carbohydrates i n the tomato plant. Plant Physiol. 24: 505-526. 83. Went, F.W. 1953. The effect of temperature on plant growth. Ann. Rev. Plant Physiol. 4: 347-362. 84. Went, F.W. 1956. The role of environment i n plant growth. Amer. Scient. 44: 378-398. 85. Went, F.W. 1957. Some theoretical aspects of effects of temperature on plants. In: Influence of temperature on biological systems. F. H. Joyson Ed. American Physiol. Soc. Inc. 86. Went, F.W. 1957. The experimental control of plant growth. Chronica Bot. Co., Waltham, 54, Mass. 87. . Wettstein, Von. 1957. Chlorophyll-letale under submikropische formueschel der plastiden. Exp. Cell.- Res. 12: 427-506. 88. Wolff, J.B. and L. Price. 1957. Terminal steps i n chlorophyll a synthesis i n higher plants. Arch. Biochem. Biophys. 72: 293-301. 5. APPENDIX I 59 Vertical gradient i n light intensity i n the Percival growth chamber. The light intensity i n the warm growth chamber was measured with a Cossen Trilux footcandle meter located at 35 cm from the front walls and 80 cm from the la t e r a l ones, at a distance from the pots as indicated below. The figures are average from three replicates. The following results were obtained: These measurements are included to better define the environmental conditions during the experiments. It must be noted that the light inten-s i t i e s to which the plants.were exposed varied with the different species since there were marked variations i n shoot structure among the species and from one developmental stage to another. Distance from the top of the pot (cm) Light . Intensity (lux) 0 10 20 30 40 50 60 . 70* 7,300 8,200 9,000 9,900 10,800 12,100 19,400 23,700 * Approximately 17 cm below the light barrier. 60 6. APPENDIX II Variations i n Chlorophyll Concentration within Plant Leaves. It was _ suspected that variations i n chlorophyll concentrations with-i n the leaves of the test species could contribute to sample error. To assess this p o s s i b i l i t y , the leaves from each of the four test species were collected from 3-day~old plants which had been grown i n the warm condition. The leaves were cut l a t e r a l l y into sections and the chlorophyl concentrations i n 0.5 g samples from these sections were determined. For Gomphrena and Phaseolus, the variations i n chlorophyll content between different leaf segments were small and were less than the differences between different leaves of the same species. The variations within leaves of Zea and Triticum were large, however, as i s indicated by the following data: Zea Chlorophyll Content (yg/gm fresh weight) Leaf Segment Leaf 1 Leaf 2 Leaf 1 230 265 230 2 534 547 530 3 828 891 820 4 1179 1059 • 1348 5 1442 1441 1453 6 1329 1356 1322 7 1290 1640 1322 8 1063 2079 1097 Leaf Total 7898 9281 7923 Average 987 1160 990 61 Triticum Chlorophyll Content (vig/gm fresh weight) Leaf Segment Leaf 1 Leaf 2 Leaf 3 1 2853 2967 2736 2 2838 2925 2700 3 2701 .2721 2654 Leaf Total 8394 8614 8090 Average 2798 ' 2871 2696 On the basis of these results, i t was decided to use entire leaves where possible, and i f this was not possible, to use leaf segments cut from the central portion of the leaf (intermediate between the leaf base and t i p ) . 62 7. APPENDIX III Determination of the Effects of Time, Temperature, and Light on the Absorbance of Chlorophyll Extracts. When chlorophyll sections are exposed to light i n the presence of oxygen, they can be irreversibly bleached. Degassed solutions or solutions kept i n darkness at room temperature do not change their absorbance. Con-sequently both oxygen and light are required for bleaching to occur (Jen, 1970; Bruinsma, 1963). Because the present experiments included four species and a large number of samples per day,, tests were done to find what storage conditions were adequate for the extracted chlorophyll samples. Chlorophyll solutions were prepared from 0.5 gm fresh weight of leaves for a l l four species. After extraction, the chlorophyll solutions were diluted to 100 ml i n volumetric flasks and kept under the conditions shown i n the table below. Absorbance measurements were made just after extraction, after 15, 30, 60 minutes, and then hourly u n t i l 8 hours after extraction. Further readings were then made daily for one month. Three replicates were made for each condition. The results are shown i n the following table. TIME AFTER. BLEACHING STORAGE CONDITION EXTRACTION % Room temperature 24 hours 20-30% and light Room temperature 1 week 0 and dark • 2 weeks 4° C and light 1 month 0 4° C and dark 1 month 0 63 From the results obtained, i t was concluded that extraction of.the samples, which took less than ten minutes, could be carried out at room temperature i f the volumetric flasks receiving the solution were covered with aluminium f o i l . For storage, the samples were therefore kept cold to prevent bleaching. 

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