THE EFFECT OF LOW TEMPERATURE AMD GIBBERELLIC ACID ON THE AMYLOLYTIC ACTIVITY AND GROWTH OF A TROPICAL GRASS (DIGITARIA DECUMBENS STENT.) By Parviz Karbassi A Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy UNIVERSITY OF FLORIDA 1971 ACKNOWLEDGMENTS The author wishes to express his deep gratitude to Professor S. H. West, chairman of the supervisory committee, and Dr. L. A. Garrard for their close and continuous supervision and patience during the course of this research and the preparation of this manuscript. Special thanks are extended to Dr. R. G. Stanley for serving * on the supervisory committee and his help, inspiration and assistance during the course of this study. The writer is also grateful to Dr. S. C. Schank, Dr. R. L. Smith and Dr. J. A. Cornell, other members of his committee, for their advice and suggestions during the course of this research. The author acknowledges with much gratitude the encouragement and help of Dr. T. E. Humphreys, Dr. G. B. Killinger and the late Dr. A. T. Wallace. Appreciation is also extended to Dr. D. E. McCloud, the chairman of the Agronomy Department, for providing an assistantship during the course of this study. ii TABLE OF CONTENTS -Page ACKNOWLEDGMENTS ii LIST OF TABLES iv LIST OF FIGURES * . v ABSTRACT vi CHAPTER I INTRODUCTION AND GENERAL LITERATURE REVIEW 1 W CHAPTER II AMYLOLYTIC ACTIVITY IN LEAVES OF TROPICAL AND TEMPERATE GRASSES 12 CHAPTER III REVERSAL OF LOW TEMPERATURE EFFECTS ON A TROPICAL PLANT BY GIBBERELLIC ACID 33 CHAPTER IV LOW NIGHT TEMPERATURE, GROWTH AND AMYLOLYTIC ACTIVITY OF TWO SPECIES OF DIGITGRASS 42 CHAPTER V SUMMARY AND CONCLUSIONS 47 LITERATURE CITED 50 BIOGRAPHICAL SKETCH 59 iii LIST OF TABLES Takle Page 1 Effect of Leaf Position on the Amylolytic Activities of Leaf Extracts of Five Weeks Regrowth of 'Pangola' 22 2 The Effect of Night Temperature Pretreat- ment and Incubation Temperatures on the Amylolytic Activities of Leaf Extracts (on Dry Weight Basis) 24 3 The Effect of Night Temperature Pretreat- ment and Incubation Temperatures on the Amylolytic Activities of Leaf Extracts (on Fresh Weight Basis) 26 * 4 Effect of Low Night Temperature and Gibberellic Acid on the Growth of 'Pangola' 39 5 Effect of Gibberellic Acid on Amylolytic Activity and Starch Content of 'Pangola' at Two Night Temperature Regimes 40 6 Effect of Gibberellic Acid on the _in vitro Activity of the Starch- degrading Enzymes of 'Pangola' 41 7 The Effect of Night Temperature Pretreat- ment on the Amylolytic Activities of Leaf Extracts of Two Species of Digitgrass 45 8 The Effect of Various Temperature Regimes on the Growth of Two Species of Digitgrass 46 iv LIST OF FIGURES Figure Page 1 Starch-degrading activity of crude extracts as shown by the starch-agar plate method. 28 2 The effect of low temperature on the amylolytic activity of 'Pangola' and orchardgrass on protein basis. 30 3 The effect of low temperature on the amylolytic activity of 'Pangola' and orchardgrass on DNA basis. 32 i v Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The Effect of Low Temperature and Gibberellic Acid on the Amylolytic Activity and Growth of a Tropical Grass (Digitar ia decumbens Stent.) Parviz Karbassi August, 1971 Chairman: S. H. West Major Department: Agronomy The potential for pasture and forage productivity of 'Pangola' (Digitaria decumbens Stent.) is markedly reduced by low night tem- peratures. Sterility and absence of variation make breeding and selection of 'Pangola' impossible. For this reason the biochemical mechanism for growth depression by low temperature and a method of reversing this effect were investigated. Growth, amylolytic activity, and the effect of gibberellic acid (GA^) on growth and certain bio- chemical events in 'Pangola' at low temperatures were determined. Crude leaf extracts were used for enzyme assay. Amylolytic activity was measured spectrophotometrically as the disappearance of starch (starch- iod ine complex). Enzyme activity varied greatly with the position of the leaves on the shoots. Young, fully expanded leaves near the shoot apices have the highest starch-degrading activity. This activity decreased in extracts of older leaves taken from progressively lower positions on the shoot. vi Amylolytic activities of leaf extracts of 'Pangola' were compared to those of orchardgrass (Dactylis glomerata L.) subjected to the same treatments. Treatment of 'Pangola,' a tropical species, with IOC night temperature for 3 nights markedly reduced the starch- degrading enzyme activity of leaf extracts whereas this was not the case with orchardgrass, a temperate species. The growth of orchard- grass is little affected by IOC night treatments. Subjecting 'Pangola' to IOC night temperature resulted in higher leaf starch content and decreased amylolytic activities, dry weights, leaf areas and shoot lengths as compared with plants exposed to 30C night temperatures. Applications of 10 5 M gibberellic acid (GA3) reversed these effects of 10C night temperature. The direct addition of GA to the reaction 3 mixture did not alter the _in vitro amylolytic activity of crude leaf extracts of 'Pangola' during their incubation either at 10C or 30C. This indicates that the effect of low temperature was on synthesis of amylolytic enzymes. A comparison was made on the growth and amylolytic activities of leaf extracts of 'Pangola' and Digitaria pentzii (P.I. 299753) at low temperatures. The D_j_ pentzii exhibits better winter survival than does 'Pangola. ' The reductions in the growth and amylolytic activity caused by low temperatures are comparable in the two digitgrass species. Data reported in these investigations suggest that reduction of growth of 'Pangola' by suboptimal night temperatures is due to the depression of amylolytic activity. There was no relation between winter survival and amylolytic activity. Moreover, the data suggest vii that treatment of 'Pangola' with GA3 may extend pasture production later into the growing season. However, further studies are necessary before it can be recommended for commercial use. viii CHAPTER I INTRODUCTION AND GENERAL LITERATURE REVIEW Introduction ' Pangola ' (Digitaria decumbens Stent., tribe Paniceae) is one of the most important pasture grasses in Florida. Over 500,000 acres of 'Pangola' are grown for pasture in the south- eastern United States (46). The major areas of distribution are in tropical and subtropical regions. Under irrigation in the Mediterranean- like climate of southwest Australia, 'Pangola' sub- stantially out-yielded legumes (70). 'Pangola,' a perennial and vegetatively propagated grass, is most suitable for use in compara- tively intensive forage systems, where forage is both grazed and harvested as hay and/or silage. The productivity and feeding value of 'Pangola' make this versatile grass an important forage crop in its area of distribution (46). However, its potential for forage productivity is reduced by low night temperatures in many areas. Sterility and absence of variation make breeding and selection of 'Pangola' impossible as methods to improve adaptation of the plant. This investigation was initiated to delineate the biochemical reasons for low temperature growth reduction. This knowledge is necessary for establishing practices which would extend the growing season of 'Pangola' in areas which experience growth reduction resulting 1 2 from low night temperatures late in the growing season. To this end a study was made of the effects of low night temperatures on 'Pangola' growth as well as on certain biochemical events. Further studies were conducted to seek a method of reversal of the effect of low temperature. General Literature Review The internal plant cell environment is constantly influenced and modified by its surroundings. Temperature is one of the most critical factors in the environment and exerts a profound effect on all physio- logical activities of the cell by controlling, among other things, the rates of chemical reactions. Since plant species differ in their tolerance to temperature extremes, some species are injured by extreme temperature while other species remain unharmed. An injury to a specific species may be caused not only by the magnitude of the variation of the temperature from an optimal value, but also by how rapidly the temperatures fluctuate as well as the length of time the temperature remains at an extreme (84) . The effects of temperature extremes on many responses of higher plants have been investigated and reviewed. Bonner (17) used the term "climatic lesion" to designate a depression in growth caused by temper- atures higher or lower than the optima for a particular plant. Plant growth includes increases in volume and dry weight (18). These are basically increases in energy found in chemical form. Growth differences reflect changes either in the photosynthetic activity of the leaf or in the rate of expansion or relative size of that photosynthetic organ (29). The final product of photosynthesis in the chloroplast of many higher plants is assimilatory starch (3, 67). Starch accumulation occurs during light periods, and during dark periods, the starch 3 may be degraded to soluble sugars and translocated to other areas of the plant to serve as respiratory substrate or in the synthesis of other organic compounds (40, 41). The level of atmospheric CO^ fixed by tropical grasses is approximately twice as great as that of temperate grasses (33). The photosynthetic efficiency of tropical grasses makes them important for increasing total forage productivity. Thus research in methods to extend the distribution of these grasses into marginal v areas has become increasingly important. Role of Temperature in the Growth of Higher Plants Growth responses of tropical and temperate grasses to tem- perature are considerably different. Many plants indigenous to tropical and subtropical areas of the world are physiologically injured when subjected to low (1 to 10C) temperatures (59). Subtropical and tropical grasses have high optimum temperatures for growth. Many species grow extremely slowly, if at all, at temperatures around 10 to 15C. In many tropical species the net photosynthetic rate remains low until 15C, and then increases rapidly up to a temperature of 35C or a little above. Temperate grasses vary little in growth response over a broad temperature range, and optimum temperatures are around 20C (29). In rice, the reported minimum temperature for germination ranges from 9 to 14C (12). Christiansen and Thomas (27) and Christiansen (26) observed reductions in plant height, fiber quality, and delayed fruiting that were directly correlated to the length of exposure of the cotton plant to cold. The duration of 10C chilling period on germinating cotton seed is additive in inhibiting subsequent seedling growth 4 at favorable temperatures. Root elongation in grasses differs in response to low temperatures. Harris (44) showed that the roots of Bromus tectorum grow well at temperatures as low as 3C, while Agropyron spicatum roots grow very slowly at temperatures below 8 to IOC. ^Beevers and Cooper (9) compared growth of ryegrass seedlings at 12C and 25C. The lower temperature reduced the relative growth rate and doubled the sugar content as a result of reduced utilization of assimilate in respiration and new growth. Reduction of growth at low night temperatures has been reported for Sorghum halepense (48) , Bouteloua gracilis . Panicum virgatum (10), and Cynodon dactylon (103). A drop in night tem- perature from 25 C to 10C reduced the growth rate of Paspalum dilatatum and Eragrostis curvula (80). Different responses have been demon- strated in tropical species. Whiteman et al. (99) showed the optimum temperatures for growth of Saccharum spontaneum to be 25C, of Sac char urn robustum to be 30C, and of Saccharum of f icinarum to be 35C. In contrast to the tropical grasses, the temperate grasses, such as Dactylis glome rata (34), Poa pratensis (76), Agrostis tenuis (63, 76), and Phleum pratense (76), have optimal temperatures ranging from 20C to 25C. The growth rate drops rapidly below 10C, but there is still some growth at 5C, and the plant remains healthy. Growth is reduced above 25C and may cease above 30C to 35C. Brown and Blazer (20) found that yields of orchardgrass (Dactylis glomerata L.) at 24 C days and 18C nights were twice those of a 35C-day/29C-night regime. 5 In temperate grasses, like tropical grasses, contrasting climatic ecotypes show differential growth rates under both low and high temperatures; a good example is Dactylis glomerata. It has been shown that the effect of temperature on the growth of orchardgrass varies according to climatic origin of the population (32, 83). Temperature effects on growth differ in different plant species. Benedict (10) showed a decrease in growth of both top and root of Bouteloua gracilis when the day temperature was 24C and the night temperature was 15C, while Agropyron smithii showed the opposite effect compared to those grown at 24C day and night temperatures. Chloroplast formation may be inhibited by low temperature, as tomatoes in a cold room at 8C have degenerate chloroplasts and abnormal grana (72). Cold treatment of 13C on Sorghum bicolor L. produced much more pigment than that of 23C treatment (75). It has been shown that mitotic cycle is temperature dependent (18,85); lower or higher than normal temperatures depress the mitotic division. Temperature influences tillering, and optimum temperature for growth varies with the species (62) . Tillering of ryegrass is increased by reducing night temperatures (4), while tillering of tall fescue is greatest when the grass is exposed to low temperatures during light periods (81). Effect of Low Temperature on Translocation and Carbohydrate Metabolism ■*' \ The transport of organic compounds from leaves to hetero- trophic cells which constitute metabolic sinks is essential to the growth of higher plants. The first responses of the plant to low 6 temperature are decreased molecular and metabolic activities, and the result of these is suppression of growth (84). Evidence has been offered to support the hypothesis that trans- location of assimilate from a leaf is dependent on factors within the leaf itself, as well as on the growth activity and assimilate utilization in other parts of the plant (89). The rate of translocation of assimilate from the leaves of sugar cane plants is more dependent on root temper- ature than shoot temperature (21). Vernon and Arnoff (87), working with soybeans, and Hartt (43), working with sugar cane, found that reduction in growth is accompanied by a reduction in the rate of assimilate translocation at low temperatures. A low temperature jacket around the hypocotyl of bean plants reduced sugar movement from the roots to the leaf immediately above the jacket (15). A fair correlation between the temperature and the rate of translocation has been shown in tomato (94). » Webb (92) studied the translocation of sugar in the primary leaf blades of Cucurb ita melopepo at temperatures ranging from 0 to 55C and found that below IOC and at 55C (and presumably above) there was an appreciable decrease in net assimilation rate; at 15C, movement of assimilate began to become restricted. Webb and Gorham (93), working with primary nodes of squash, found a zero rate of translocation at OC or 55C and the highest rate at 25C. Geiger (39) demonstrated that little translocation occurred when the translocation path of sugar beet plant was subjected to 2-5C temperature treatments, relative to trans- location rates at 28C to 30C. Temperature has a large influence on the amount of organic acids and sugars in the plant (56). There is evidence that accumulation of assimilates in a leaf causes a decrease in the rate of assimi- lation in that leaf. In 1868, Boussingault proposed that the accumu- 7 lation of assimilates in an illuminated leaf may be responsible for a change in the net photosynthetic rate of that leaf (8). Kostytschew et al . (52) stated that midday depression of photosynthesis is caused by assimilate accumulation in the leaves. In an excellent review article, Neales and Incoll (65) reported that leaf assimilate levels may control the rates of photosynthesis and growth. Curtis (30) demonstrated that lowering petiole temperature of bean plants to between 1C and 4-6C increased the carbohydrate content of the leaves and effectively retarded translocation. Burt (23) lowered the net leaf assimilation rate of potato plants by reducing the air temperature around the potato tubers. Thorn and Evan (82), Humpheries (47) and Burt (22) suggest that temperature may partially govern the photosynthetic rate of the plant by varying the ability of the plant to utilize the assimilate. Warren(90 }91 ) concluded that the depression of net assimilation rates in arctic regions is due to low temperatures which cause sugars to accumulate to levels at which they depress assimilation. Cell membrane permeability changes and lateral release of assimilates could explain some of the temperature effects on translocation (89). Amylolytic Activity in Plants Amylases are found in almost all plant cells. Alpha- amylase, a calcium-requiring enzyme (104), attacks starch randomly at ot( 1*4) linkages, and beta-amylase hydrolizes starch, starting at the non-reducing ends of the chain, splitting off beta-maltose by breaking the oL. (1-4) linkage (78). Together they liberate a mixture of re- ducing sugars. Both enzymes attack amylose and amylopectin. In 8 the presence of inorganic phosphate, phosphorylase breaks down starch and liberates glucose-l-phosphate from non-reducing ends of starch molecules (3) . In 1890, Haberlandt (42) observed the liquefaction of the starchy endosperm portion of rye seeds and the dissolution of starch grains by substances produced in the aleurone layers. It is now established that alpha-amylase is the chief enzyme involved in the initial degradation of starch in storage tissues to more soluble forms (11, 79), while phosphorylase and beta-amylase assist in the further conversion of these forms to free sugar (11). Also, phos- phorylase (31), alpha-amylase (38), and beta-amylase (40, 41) are reported to be responsible for degradation of starch in the leaf plastids. Changes in the environment of these enzymes will alter their behaviors and functions. It is often found that at temperatures well below optimum, Arrhenius' equation (plotting the log of velocity against the reciprocal of absolute temperature) no longer applies. However, an apparent activation energy higher than expected occurs at these temperatures (74) . Resistance to this type of low temperature inhibition might be effected by an increased production of the rate- limiting enzyme and/or a change in the level of activation energy required. Walker and Hope (88) showed that as the temperature is increased, in addition to the expected activation of alpha- amylase, an effect was noted in the hydrolysis of raw starch granules. They worked with saliva and crystalline salivary alpha-amylase at low temperatures and showed that the extent of adsorption to starch granules increased at low temperatures and higher pH's. If conditions 9 unfavorable for the adsorption of the enzyme were chosen, the rate of hydrolysis of starch granules was increased. According to Badenhuizen (7) spaces between starch molecules are different in starch granules from various sources. Therefore, the action of amylolytic enzymes may take place as exocorrosion or endocorrosion, e.g.j acting on the outside of the starch granules or penetrating into the interior of the granules, depending on the nature of the starch molecules. Thus the substrate can be an important factor for determining amylolytic enzyme activity. Large differences have been shown in the required activation energies for the same enzyme when it is isolated from different sources. Activation energy levels (in K cal/mole) reported for alpha-amylase are as follows: 7.0 in Hordeum, 10.5 in Aspergillus, and 13.5 in Sus sp. (36). Effect of Growth Regulators on the Amylolytic Activity and Growth of Plants The causes of low temperature lesions are still not known for certain, but a theoretical basis exists as a result of recent studies on enzyme behavior at low temperatures (57). It has been stated that depression of growth by low temperature may be overcome by production of a higher concentration of the rate-limiting enzyme(s) Ketellapper (54) demonstrated that the adverse effects of unfavorable temperature on plant growth can be partially or completely prevented by application of essential metabolites to plants growing in such unfavorable conditions. The nature of the effective- metabolites depended on the plant species involved and the temperature at which it was growing. When thiamin was applied to Cosmos at temperature 10 regimes of 17C days and 10C nights or of 20C days and 14C nights, a 30% increase in dry weight resulted (16, 53). Application of a 10% sucrose solution to pea plants grown at 23C days and 17C nights caused a 56% increase in dry weight bringing the dry weight up to the level of plants grown under optimal conditions of 17C days and 17C nights (53). Attempts to increase the yield of annual crops such as Triticum vulgar e and Zea mays by application of gibberellin have been unsuccessful (2, 64 ). In contrast, it has been shown that Poa sp. and Cynodon dactylon responded more to gibberellin at low temperatures (101). The most pronounced effect of gibberellic acid is modification cf plant growth. Gibberellic acid (GA) has been shown to increase alpha-amylase activity in barley endosperm (28, 35, 49, 55, 66, 86, 102). Protein and ribonucleic acid can be regulated by GA (37). Studies (25) with aleurone cells of barley indicate that GA^ enhances the incorporation of ^C-uridine and ^C- adenosine into RNA, and the authors concluded that gibberellin stimulates de nova synthesis of alpha-amylase by controlling the synthesis of mRNA. GA accelerated growth and amylase activity in germinating barley seed; this effect is inhibited by chloramphenical sodium succinate, indicating that alpha-amylase is synthesized during germination and that GA accelerates this synthesis (77). Application of exogenous GA^ to a clone of Trifolium pratense caused the formation of two new protein peaks four and fifteen days after the treatment (50) . Treatment of pea plants with GA^ markedly increased protein synthesis (19). Blacklow and McGuire (13) have shown that a foliar spray of 1% GA increased foliage growth of tall 11 fescue during the winter. GA enhanced tomato fruit ripening at both higher and lower than optimal temperatures (1). Extension of wheat-leaf sections by GA^ application has been shown (68). It seems that the endogenous level of GA^ or a block in the utilization of GA- would determine the effect of applied GA„ . Radley (69), working with tall and dwarf wheat, found that application °f GA^ to tall wheat cultivars markedly stimulated the growth of tall seedlings but not the growth of dwarf seedlings. GA^ increased the amount of soluble carbohydrates in the leaves of the tall cultivars but not in those of the dwarfs. A higher amount of endogenous GA^ occurred in the germinating grains, light-grown seedlings, and developing stems of dwarf cultivars than in those of the tall cultivars. In seeking a solution, first the effect of low temperatures on the starch-degrading enzymes of temperate and tropical plants was investigated. These two types of plants have different growth optima regarding temperature. Therefore, a comparative study was made on the effect of temperature on the amylolytic activities of crude leaf extracts. Comparisons were made between tropical 'Pangola' (Digitaria decumbens Stent.) and temperate orchardgrass (Dactylis glomerata) . CHAPTER II AMYLOLYTIC ACTIVITY IN LEAVES OF TROPICAL AND TEMPERATE GRASSES Introduction Growth and photosynthesis of 'Pangola' (Digitaria decumbens Stent.), a tropical grass, are reduced by night temperature of IOC (14, 61, 95, 96, 97). Studies of leaf ultrastructure, as well as chemical analyses, showed that illumination of the plants for 12 hr at 30C resulted in the accumulation of large amounts of starch in the me sophy 11 chlorop lasts . This starch disappeared from the chloro- plasts when the plants subsequently were placed in darkness for 12 hr at 30C. In contrast, accumulated starch did not disappear from the chloroplasts during a 12-hr dark period of IOC (45). In orchardgrass (Dactylis glomerata L. ), a temperate grass, the growth of which is not severely retarded by IOC night temperature, there was also an accumulation of starch in chloroplasts during illumination at 30C; however, this starch was mobilized and translocated from the chloroplasts during the dark period even at temperatures as low as IOC (98). Reduced growth of 'Pangola' may be caused by the failure of starch to be mobilized and translocated from the mesophyll chloro- plasts during periods of low night temperature and, as suggested by Hilliard and West (45), this may interfere with photosynthesis. West (97) observed a reduction in the photosynthetic capacity of 12 13 plants subjected to low night temperature as evidenced by the reduced ability of chloroplasts from IOC-treated plants to perform the Hill reaction and by the increased packing volume of these organelles. Much evidence now supports the concept that leaf assimi- late level is a controlling factor in the rate of photosynthesis (65). The failure of accumulated carbohydrate to be translocated from the chloroplasts of 'Pangola1 may indicate that some temperature- dependent limitation affected starch-degrading enzymes. For this reason, a comparative study of the effect of temperature on amylolytic activities of leaf extracts of 'Pangola' and orchardgrass was undertaken. Materials and Methods Plant Materials 'Pangola' plants were propagated from cuttings and potted in 10-cm plastic pots containing a mixture of peat and sterilized (steam- treated) potting soil (1 : 1). Orchardgrass plants propagated by division were potted similarly. All plants were maintained in a greenhouse with a 27C minimum temperature and no supplemental lighting. The plants were watered every other day and received weekly applications of liquid fertilizer. In 'Pangola, ' the plant top growth was cut back periodically to approximately 5 cm to maintain the plants in a rapid vegetative growth stage. Temperature Pretreatments After 5 weeks of regrowth, 'Pangola' and orchardgrass plants were subjected to two pretreatment regimes for a period of 3 consecutive 14 days and nights. During the day, all plants received illumination of 3000 ft-c for 12 hrs at 30C, whereas during the 12-hr night period, they were held in growth chambers either at 10C or 30C. These plants subsequently were used for the preparation of crude enzyme extracts, determination of protein and DNA content, and determination of fresh and dry weights. Preparation of Enzyme Extracts Crude enzyme extracts were prepared by grinding 3.0 g of young, fully expanded leaves in an Omni-mixer with 24 ml of ice-cold CaCl2 (0.001 M). The homogenate was allowed to stand for 1 hr in an ice bath and then centrifuged at 27000 x g for 30 min. The supernatant fraction was passed through Whatman No. 2 filter paper; the filtrate was used immediately as the enzyme source. Qualitative Determination of Total Amylolytic Activity of 'Pangola' A modification of the starch-agar plate method of Gates and Simpson (38) was used to determine qualitatively the amylolytic activity of 'Pangola' extracts. The media for starch-agar plates consisted of 2 g agar, 1 g amylose.O.l g CaCl2, andO.l g KH2P04 in 100 ml acetate buffer pH = 5.2. Filter paper was placed on top of jelled media in petri plates, and enzyme extracts were pipetted on the filter paper. Amylolytic activity was measured by the differential iodine staining of media after incubation at 40C for 24 hrs. Quantitative Measurement of Amylolytic Activity Amylolytic activity was determined by measuring the rate of disappearance of iodine-staining starch during a period of incubation 15 with a portion of the crude enzyme extract. The starch substrate for this reaction was prepared by the addition of 5.0 g of soluble starch and 2.7 g KH2P0^ to 500 ml of distilled H^O. The solution was boiled for 1 min, cooled to room temperature and passed through Whatman No. 2 filter paper. Iodine solution was prepared just before each experiment. Five ml of a stock solution (3.0 g KI and 1.27 g I2 per liter) was diluted to 200 ml with 0.2 M HC1. This diluted solution was used directly in the assay. The enzyme^ reaction mixture for 'Pangola' contained 4.0 ml of succinate buffer (0.05 M, pH 5.2), 2.0 ml of starch solution and 4.0 ml of the crude enzyme extract. In case of orchardgrass , 6.0 ml of succinate buffer, 2.0 ml of starch solution and 2.0 ml of crude enzyme were added to the 25-ml test tube. Three incubation tem- peratures were used (incubation being the period of _in vitro enzyme- substrate reaction): 10, 20 and 30C. At a predetermined time, 1.5 ml of aliquots were removed from the reaction test tubes and were mixed with 5.0 ml of I^HCl solution. The absorbance of the starch- iodine complex was determined spectrophotometrically at 625 nm. Appropriate zero-time controls were used so that enzyme activity could be expressed as the change in absorbance during the incubation period. Protein Determination » One ml 10% TCA (trichloracetic acid) was added to an equal volume of crude enzyme extract, and the mixture was chilled in an ice bath for 1 hr. The precipitate was separated by centrifuging 20 min at 20000 x g at 0C. The precipitated protein was hydrolyzed with 1 ml of 1 M NaOH for 24 hr at room temperature. Protein was determined by the method of Lowry et al. (60). 16 DNA Measurement Three g of 'Pangola' leaves or 1.5 g of orchardgrass leaves were ground with a 5 % sucrose solution containing 0.01 M tris, 0.01 M MgC^, and 0.06 M KC1 in an Omni-mixer for 5 min at 0C. The homogenate was centrifuged at 5000 x g and the supernatant passing through a glasswool pad was used for DNA determination. DNA was determined by a modification (73) of the Burton method (24). To 4 ml of the above leaf homogenate an equal volume of 1.0 M HC 10^ was added, and the mixture after 1 hr in an ice bath was centrifuged 20 min at 27000 x g. After the supernatant was discarded, the precipitate was washed with a mixture of ether, ethanol, and chloroform (2: 2: 1) to remove lipids and chlorophylls. The wash solution was then centrifuged for 20 min at 30000 x g. The precipitates were hydrolyzed with 2 ml 0.6 M KOH for 16-20hr. After this time, 6 ml 0.5 M i£10^ were added to cause reprecipitation of DNA, and the suspension was centrifuged 20 min at 27000 x g at > 0C. After adding 4 ml hot 0.5 M HCIO^ to the precipitate, it was incubated at 70 to 90C for 1 hr. This was centrifuged 20 min at 27000 x g, then 8 ml of d iphenylamine reagent were added to 4 ml of supernatant. This was boiled for 15-20 min, then centrifuged at 12000 x g. The absorbance of the supernatant (containing DNA) was determined at 600 nm. Absorbance for each sample was compared to readings of similarly prepared calf thymus DNA standards (24). 17 Results and Discussion Qualitative Determination of Starch-degrading Enzyme of 'Pangola' In these studies, the term "amylolytic activity" is used to refer to the starch breakdown without indicating the involvement of any specific enzyme, for it is not known specifically which starch- degrading enzymes are active in the crude leaf extracts. Determination of the amylolytic activity by the starch-agar plate method did not disclose differences between extracts from IOC or 30C pretreated plants. Starch hydrolysis was found in the case of both treatments (Fig. 1). This procedure was not sensitive enough to be used to comparing treatments, therefore, subsequent data are from the starch- iodine method of determination. The starch-agar plate method, however, is a simple method which may have application to some worker for screening or selection for breeding. Effect of Leaf Position on the Amylolytic Activities of Leaf Extracts In previous studies, much variation was encountered in the starch-degrading activities of leaf extracts among replicates from the same temperature treatment. To determine some of the factors responsible for this variation, the effect of leaf position on the amylolytic activities of leaf extracts was studied. Starting with the first fully expanded leaf at the vegetative shoot apex, the leaves were numbered consecutively down the shoots. The first, second, third and fourth leaves were taken from 10 uniform plants of 5 weeks regrowth at 30C, and crude enzyme extracts were prepared. The highest starch-degrading activity was found in the extracts of 18 the youngest or uppermost leaves, and this activity decreased as the leaves were taken from progressively lower positions on the shoots (Table 1). This finding is consistent, perhaps, with the idea that upper leaves are in a favored position for the reception of light and thus may produce larger amounts of starch. In addition, those younger leaves are proximate to and supply assimilate for a most active metabolic sink, the rapidly growing apical region of the shoot. The presence of this sink for leaf assimilate would also favor comparatively high rates of carbohydrate production in the upper- most leaves (89). If, indeed, the rates of carbon assimilation in young leaves are sufficiently high to raise the levels of assimilate starch, it is likely that greater amounts of starch-degrading enzymes would also be present. Data of Table 1 also show the importance of judicial leaf selection in preparing extracts for the determination of amylolytic activity. In the following experiments, amylolytic activities were determined by using leaf samples combining both the first and second fully expanded leaves of uniform shoots. Effects of Low Temperatures on the Amylolytic Activity Pretreatment of 'Pangola' at a night temperature of IOC reduced the amylolytic activities of leaf extracts when compared with the activities of extracts from plants receiving 30C nights (Table 1) . This reduction in enzyme activity occurred irrespective of the incubation temperature. For incubation temperatures at 10, 20 and 30C, the reductions in amylolytic activity (on dry weight basis) 'attributable to 10C night pretreatment were 53%, 51% and 47%, respec- 19 tively. In contrast, pretreatment of orchardgrass at IOC night temperature had no effect on the amylolytic activities of leaf extracts. This was true at all incubation temperatures. The temperature during enzyme assay had a pronounced effect on enzyme activity irrespective of pretreatment and species. However, the starch-degrading activities of 'Pangola' extracts were reduced to a greater extent by low temperature than the activities of leaf extracts of orchardgrass. In 30C pretreated 'Pangola' changing the incubation temperature from 30C to IOC reduced the amylolytic activity of 'Pangola' extracts by 69%, whereas the activity in orchardgrass extracts were reduced by only 52%. Similar reductions were observed using extracts from IOC pretreated plant material. Amylolytic activities of the leaf extracts from the various treatments were also calculated on the basis of activity per unit protein (Fig. 2), activity per unit DNA (per cell basis) (Fig. 3), and activity per unit fresh weight (Table 3). In each case, results were the same as those based on activity per unit dry weight (Table 2). Low night temperatures depress growth in 'Pangola'; this growth depression is accompanied by an accumulation of starch in the meso- phyll chloroplasts (45). West (96) stated that accumulation of starch may result from: 1) increased synthesis of starch, 2) decreased activity of starch-degrading enzymes, 3) decreased utilization of the products of starch degradation at metabolic sinks, and/or 4) a decrease in translocation of the products of starch breakdown. In the present work one of these proposed causes, the effect of low night temperature pretreatments on starch-degrading activities of crude leaf extracts from 'Pangola,' has been investigated. Further, as 20 a comparison, the effects of low night temperature pretreatments on the starch-degrading enzymes of orchardgrass was determined. The effect of temperature treatments during incubation on the starch- degrading activities of leaf extracts of 'Pangola' and orchardgrass was also determined. Alpha-amylase, beta-amylase, and phosphorylase are generally believed to be the enzymes responsible for starch degradation (3). But there is much confusion as to the localization and compart- mentation of these enzymes in relation to their function in the plant fb was assumed that in the main. This method measured activities of the amylases. Since preliminary experiments did not demonstrate appreciable phosphorylase activity, assay conditions favorable for amylase activity were used routinely. Both alpha- and beta- amylases have been postulated to have roles in starch degradation in leaf tissue. Haapala (41) attributed the rapid hydrolysis of starch accumulated in chloroplasts of Stellaria media during prolonged illumination to a rapid increase in the activity of beta-amylase. Gates and Simpson (38) found an association between the starch contents and alpha-amylase activities of leaves of a wide range of higher plants. Further, Juliano and Varner (51) suggested that alpha- amylase is the major enzyme involved in the initial degradation of starch of pea cotyledons to more soluble forms and that beta-amylase and phosphorylase assist in the conversion to free sugars. In brief, it has been shown that 10C night temperature pre- treatment strongly repressed the starch-degrading activity of extracts of Pangola leaves. This was not the case with leaf extracts of orchard grass. Low temperatures of incubation during enzyme assay decreased 21 the activities in extracts from leaves of both 'Pangola' and orchard- grass; however, the effect on the tropical species was more pronounced. These results explain, at least in part, the preferential accumulation of starch in the chloroplasts of tropical grasses during periods of low night temperature and, consequently, reduced photosynthesis and growth. West (96) suggested that the accumulation of starch in chloroplasts when plants were subjected to IOC night temperature provided evidence for any of several mechanisms for the influence of low temperatures on growth and photosynthesis: 1) the accumulation of starch allows a concomitant accumulation of certain photosynthetic intermediates which by their presence inhibit further CO2 fixation, 2) continued accumulation of starch without degradation and transport might fill the chloroplasts to the extent that they are injured physi- cally, thus impairing their function, and 3) the starch grains them- selves might actually provide a shading effect on the chloroplasts below, thus preventing efficient light utilization. These results would equally as well explain the resistance of temperate grasses to growth retardation by low night temperatures. In the latter case, starch-degrading enzymes would remain active to produce the soluble sugars which are characteristically found in leaves of temperate plants during periods of depressed temperature. Table 1. EFFECT OF LEAF POSITION ON THE AMYLOLYTIC ACTIVITIES OF LEAF EXTRACTS OF FIVE WEEKS REGROWTH OF 'PANGOLA' Leaf position AMYLOLYTIC ACTIVITY (apex to base) (AOD/g fr. wt./25 min) 1 5.72 2 4.61 3 3.09 ' 4 2.27 The activities of leaf extracts were determined at 30C. Leaves were numbered consecutively from the uppermost fully expanded leaf of the shoot to the base of the shoot. F test shows a significant difference at P = .01. *Each entry in Table 2 is an average of three measurements. Individual values 4-J d CO o o d O d •H d *3 4-1 CD o d E U •3 ix d o rQ 00 d in •H d 3 •r-4 rd > d o CO d o d o • rC •H •r-4 o 4-J 4-4 r— 1 00 CO CJ •H CD rd d 3 CO d J-4 CD 4-J rd CD r— 4 d d 4-1 r-4 E d CD 3 3 3 u E d d E 4-J CO IX ,C CO cj o • i—4 4J CO 44 3 O 4-1 3 3 J-4 00 3 d 4J d d J-4 p 3 3 p t—4 E P 3 •r-4 4-J 4-J r-4 d 3 o E •r-4 4-J CO d — rQ d 3 d o o d d •H 3 Q rd 4-J O •r-i 4-1 £ 00 CO d 4-» •H d 3 3 J-4 E O •rl O u o 3 d P X in 3 o • 4-1 o O II CO P d d 4-J 3 d E u 3 4-4 4-i •H 3 3 •rl u u o H M. *3 3 d d « P d P • J-4 p O • rs VO i—4 £ m 3 3 • 3 r—4 4-J CO ft II • IX d O •r-4 d CO 3 M 3 r» • d r—4 4-J •H i—4 CO d •H 3 4-J X 4-J rd 1 o £ 3 d 00 3 p d rd o d o p d £ d 3 o r-4 • P CO CO •H 3 o 4-J J-4 •H r-4 3 4-J 3 3 CO E 3 •r-4 O 4-J CO o d — J-4 4-J d P CO d o 4-J 4-4 d CO O 3 3 Q 4-J CO 3 O 3 4-J 00 3 d 3 00 d 3 d u O •r-4 o *3 3 u J-4 r—4 p O Cu 3 •r-4 3 O 4-J d d r-4 •d 3 *n E C0 o 3 • CO r—4 o — P II d •H d O p o d d •r-4 4-J 3 u d Q p u 3 m •r-4 3 o vD Table 2. THE EFFECT OF NIGHT TEMPERATURE PRETREATMENT AND INCUBATION TEMPERATURES ON THE AMYLOLYTIC ACTIVITIES OF LEAF EXTRACTS (ON DRY WEIGHT BASIS) 24 1 o 4* O CM d •H G vO CO vO O ■u G 00 • • d d av CM CO co •4* co vO r— 1 CO CTv •>< d r-l CM VO vO T— 1 in 4 r-l co CM >-> •!-! 4^ E •l-l CM in CO CN r^. CA VO o CO CM CO 4* > o VO *4- co n vO vo 00 CM O r-l OV •H X x CO , X \ d vO rH VO r— 1 4 00 4* r—i n 00 o x < £ CD e r— i co VO vO CM •4- •4- r— 1 CM CM u d (D CO o CO 4 VO r— 1 n CO r-l VO 00 4" ■u no a VO in CM 4 4 00 r— 1 00 00 o vo X CM H o" m o CM co CT\ CO o m co r- CO O ^ r Q O CM >4 r— 1 r— 1 4 m I— 1 CM cn E <3 < U0 n ov r— 1 VO r— 1 00 VO crv 00 CM co vO 00 vo vO vO c vo CO CM CM CM CM T— 1 m crv r— 1 m r— 1 vo in CM •4- r^« r— 1 CM 4 in r— 1 CM cn 4-1 d E u CD p-i H o o r-H co O O rH CO o o r-l CO o o r-l CO o o r-l CO o o i-i co CO to G u o d d r-l nj r— 1 O d o 00 d 00 d X d d o d pm M PM - O d nj r— 1 no U o u d 00 d X d X o d o d P-t o - o a d d o d ♦r-l X 4-1 d 4-1 • 4-J • £ •rl •r-4 d CM CM vO 4-J 4-4 •H 60 CO 60 CM 0 d •rl > d d • o P d d •r4 •r-4 d d 60 rd II > II > 11 d o •r4 d d o d CO CJ IX rd x .e 1 •r4 •rl o CO CO CO 4-1 Po CO o a •rl CD r-4 o o d rd rd 4-4 • CM • r-4 • 60 4-J 60 O 4-1 4-J 4-1 •H •r4 CO 4-4 CO 4-4 CO CO 4l rd CD CD O 0 O 0 o 44 4-1 4-1 4-1 'O 4-t d d 0 0 d 4-J CD 4j 0 44 0 d IX CD d 60 d 60 d 60 CO > 44 d 4-J d 4-J cs r\ d CD d d d d d 0 rd Oh 44 44 44 44 44 U • £ 0 0 d d o CD av vo p CO O o r-4 G G 3-4 (dj CO VO CN CN i—4 CO oo O'* r—4 CN U0 VO P-4 r— i r—4 T— 4 •H 4-) CO VO o 00 UO oo 00 00 00 p 4-J 4J d co VO r— 4 co r—4 CO 00 Ov r—4 <3* iO y is 0) i — 1 < e rC •p4 O co u •H G G vD co CN p P r—4 U0 r-4 G\ 4-J 3-4 Pp Ph & CN ON CN P o UO r-4 00 r—4 r—4 o o o r— 4 w CO p* CN CO CN CO p ON r—4 CN LO p O 60 r— 4 r—4 t— 4 >. Q E O < A co r—4 CN p co P p av o U0 ov co r-4 CO O sf CP 00 co o 00 P t— 4 • • • • • • • • • • • • co p. co co cn -* 4J 4-J PQ CJ rC 00 00 •H •H § CJ CJ O PC CO CO CO Cl Ci CO o o < 4H 4H CO CJ CJ H O O cj CO r— 1 c§ 4-J 4J H cd cd X w TJ o o I M 00 00 Ci cd H CJ c o Jd CJ cd cd 4-J PC < PC CC cd i—4 - — PS < o z M • • • • P r— 1 CSJ CO <1* 0) U P 00 28 n 2 < 3 O U 2 < Pm O H U < u M H J>m O (J CSJ (U M 3 60 •H Pm pcn2ola ORCHARD-GRASS 30 o o CM O ro INCUBATION TEMPERATURE AMYLOLY l 1C ACTIVITY (AOD/mg profem/IOmin) X 10 ESSS3 30°c PRETREATED ' 1^3 I0°c PRETREATED < o o 2 < H W CO £j < S pq W g° l-J ^ o O CO CO E-< O M w o pH Q Eh 2 “S w o K Pi H O co 30C night temperature. 47 48 Application of GA^ reversed the effect of low night tempera- ture on the growth of 'Pangola. 1 This was evidenced by increases in dry weight, leaf area, and shoot length. In addition, treatment with GA^ increased the amylolytic activity in crude leaf extracts and decreased the level of accumulated starch in leaves of plants subjected to IOC for three nights compared to plants subjected to IOC nights without GA^ treatments. Direct addition of GA^ to the reaction mixture did not alter in vitro amylolytic activity of crude leaf extracts of 'Pangola' during incubation at IOC or 30C. This observation supports the idea that low temperature effects were due to decreased synthesis of amylolytic enzymes, not allosteric interaction of GA^ with the enzyme. A comparison was made of the growth and amylolytic activi- ties of leaf extracts of 'Pangola' and Digitaria pentzii (PI 299753) at low temperatures. Eb_ pentzii exhibits better winter survival (as distinguished from the effects of low temperature) than does 'Pangola.' The reduction in the growth and amylolytic activity caused by low temperatures are comparable in the two digitgrass species. 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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. S. F. West, Chairman Professor of Agronomy I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. try and Botany I certify that 1 have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. S. C. Schank Associate Professor of Agronomy I certify that I have read this study and that in my opinion conforms to acceptable standards of scholarly presentation and fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. it is JL R. L. Smith Associate Professor of Agronomy I certlfy that I have read this study and that in my opinion conforms to acceptable standards of scholarly presentation and ully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. it is J. A. Cornell Assistant Professor of Statistic s ,T^iS f isser tat ion was submitted to the Dean of the College of Agriculture 1 ° !C Gradua^e Council5 and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. August, 1971 Dean, Graduate School