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Chemical and biochemical responses of sugar beet root to foliar freezing and defoliation White, Gordon Allen 1955

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CHEMICAL AND BIOCHEMICAL RESPONSES OP SUGAR BEET ROOT TO FOLIAR FREEZING AND DEFOLIATION by GORDON ALLAN WHITE A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n the Department of BIOLOGY AND BOTANY We accept t h i s thesis as conforming to the standard required from candidates f o r the degree^of MASTER OF ARTS Member^ of the Department of BIOLOGY AND BOTANY THE UNIVERSITY OF BRITISH COLUMBIA October, 1955 ABSTRACT SOME CHEMICAL AND BIOCHEMICAL RESPONSES OF SUGAH BEET ROOT TO FOLIAR FREEZING AND DEFOLIATION Sugar beet seed, S.K.E.-R-ll, was obtained from the B.C. Sugar Co. Ltd., Vancouver, B.C. and germinated i n f l a t s i n a greenhouse on January 29, 1954. The beet plants were transplanted to a f e r t i l i z e d f i e l d on May 2, 1954. A randomized l o t design was chosen i n order to reduce er r o r caused by s o i l d i f f e r e n c e s , moisture v a r i a t i o n s , and pH e t c . T h i r t y groups of 10 beets per group were selected from the randomized l o t . The leaves of 6 groups were frozen with dry ice and the other groups were d e f o l i a t e d , decrowned continuously d e f o l i a t e d , or used as controls." The regrowth on the continuously d e f o l i a t e d beets was removed every two days following i n i t i a l d e f o l i a t i o n . D e f o l i a t i o n was e f f e c t e d by s l i c i n g o f f the leaves one-quarter inch above the crown. Decrowning was done by c u t t i n g the beet root transversely j u s t beneath the outer r i n g of meristematic buds. The: d e f o l i a t e d beets were used to serve as a p a r a l l e l to the destruction of leaves by freezing. The continuously d e f o l i a t e d beets were a check on the d e f o l i a t e d beets, where i t was considered that photosynthesis i n the new regrowth leaves would p a r t i a l l y o f f s e t a large sugar l o s s i n the root. Two experiments were completed. The f i r s t experiment and treatment began on August 16, 1954; the second on October 13, 1954. Haivest times were at the 1, 4, 8, 11, 16 and 20 day i n t e r v a l s following Aug. 16, and at the 1, 4, 8, 12 and 15 day i n t e r v a l s following October 13. Enzyme a c t i v i t y only was determined i n the second experiment. 'The fresh l e a f weights of the d e f o l i a t e d and control beets were recorded and l a t e r compared with l e a f regrowth weights and sugar content. The beets were harvested i n groups of 10 beets a l l t r e a t e d i n one s p e c i f i c manner. Ten beets of each group were removed from the s o i l and each beet s l i c e d diagonally across the centre region. The sections were washed i n water and pulped i n a meat grinder g i v i n g approximately 2000 grams of pulp from 10 sections. Three hundred grams of pulp was used in dry weight -determination. Forty grams of fresh pulp from each group was blended f o r 2 minutes with 100 ml. of. d i s t i l l e d ice water i n a Waring blendor. The sol u t i o n was f i l t e r e d through broadcloth and used i n enzyme a c t i v i t y measurements. In the second experiment, 47 grams of pulp was blended with 100 ml. of d i s t i l l e d ice water f o r 2 minutes. The crown portion of the root was used i n the estimation of invertase a c t i v i t y . A check on the sampling method showed that the 40-gram aliquot o f pulp used f o r enzyme determination represented the sample. Sucrose percent and phosphatase a c t i v i t y were used as the basis o f t h i s t e s t . The f r e s h pulp was analyzed f o r sucrose, invert sugars, dry weight, catalase, phosphorylase, beta-amylase and invertase enzyme a c t i v i t i e s . The dri e d pulp was ground to 40-mesh and analyzed for t o t a l nitrogen, sucrose and invert sugars. Insoluble nitrogen and starch-dextrins were determined i n ethanol extracted pulp. Duplicate determinations were made on each sample. Percentages are based on both dry and fresh weights and given as T/C values. Phosphorylase, phosphatase, catalase, beta-amylase, invertase were measured. Sucrose, invert sugars, starch-dextrin3, and t o t a l and insoluble nitrogen were also determined. The.highest amount of l e a f regrowth occurred 4 - 1 7 days a f t e r freezing. The r e s u l t s i n d i c a t e d no r e l a t i o n between l e a f weights and sucrose content nor between root weight and sugar content i n mature beets. The percent•dry weight•decreased i n a l l treated beets from the 1st to the 20th days a f t e r treatment. This decrease i s l i k e l y a r e s u l t of sucrose l o s s and an increased hydration i n the beet root. Sucrose percent based on dry and fresh weight generally decreased following a l l - treatments. A p o s i t i v e c o r r e l a t i o n between percent sugar l o s s and l e a f regrowth i s suggested. There was an increase i n the amount of reducing sugars a f t e r f o l i a r l o s s . The suggestion has been made that the monosaccharide sugars are u t i l i z e d almost immediately i n l e a f regrowth or i n (increased r e s p i r a t i o n i n the beet crown. The percent, of starch-dextrins tended to decrease i n the treated beets but t h i s i s most l i k e l y not s i g n i f i c a n t . The decrease i n percent of t o t a l carbohydrates found follows the fact that sucrose disappears. To t a l carbohydrate estimations seem to provide a reasonable basis for determining the amount of sucrose los's. T o t a l and soluble nitrogen values decreased to the 8th day a f t e r treatment and increased a f t e r t h i s time. Insoluble nitrogen r e s u l t s were generally inconclusive. The r e s u l t s suggested a t r a n s l o c a t i o n of soluble nitrogenous compounds to the beet crown where active growth was occurring. The apparent a c t i v i t y of phosphorylase decreased with time in a l l treatments. Starch phosphorylase i n sugar beet root l i k e l y has a minor role i n t o t a l carbohydrate metabolism of the t i s s u e . Phosphatase a c t i v i t y decreased to -the 11th day i n every treatment except decrowned. The reason for a lower apparent phosphatase a c t i v i t y in treated beets i n t h i s experiment i s not known. It may be associated with an increase r e s p i r a t o r y r a t e . There were no s i g n i f i c a n t changes i n beta-amylase a c t i v i t y and no c o r r e l a t i o n could be found between starch-dextrin content and amylase a c t i v i t y . Catalase a c t i v i t y based on monomolecular values, decreased with time a f t e r treatment. A decrease i n catalase a c t i v i t y might be expected i n the mature, c e l l s o f the root as the r e s p i r a t i o n rate decreases with age, A c o r r e l a t i o n between invertase a c t i v i t y and sucrose l o s s was indicated i n the frozen and decrowned beets but not i n the d e f o l i a t e d beets. From the r e s u l t s of t h i s experiment i t seems u n l i k e l y that invertase i s alone responsible f o r a sucrose decrease. The r e s u l t s found i n t h i s experiment were l a r g e l y negative. X CHEMICAL AND BIOCHEMICAL RESPONSES OF SUGAR BEET ROOT TO FOLIAR FREEZING AND DEFOLIATION* 1. INTRODUCTION The experiment undertaken here arose d i r e c t l y from the concern of Canadian sugar beet technologists over sugar l o s s incurred by early f a l l f r o s t s before and during sugar beet harvest i n southern Alberta, Canada* This p a r t i c u l a r phase of sugar beet physiology research stemmed from a con* ference on phys i o l o g i c a l research problems In the sugar beet industry, held at the Dominion Experimental s t a t i o n , Leth-bridge. Alberta on May 8, 1952* At t h i s conference i t was resolved that a research program be i n i t i a t e d f o r the study of basic plant physiological problems i n connection with the production of sugar beets* I t was also resolved that such a program should include research concerning the e f f e c t s of f r o s t on growth and sugar content of sugar beets* sugar losses during p i l e storage of beets were discussed at the conference* I t has been known f o r some time that one e f f e c t of autumn f r o s t s on sugar be,©ts i s the decline i n growth and sucrose content; of the beet root. I t i s considered that a 2. a temperature of 6°p w i l l cause k i l l i n g of most of the tops and do some crown injury* Data have shown that the sugar content of beets tends to rise u n t i l harvest time unless a 26°p frost ooeurs* The sugar content i s usually decreased following the f i r s t frost, regardless of i t s intensity below 26°p. AS this decrease was found to appear within five days, i t was not associated with obvious leaf regrowtiuU). The percent sucrose reduction i s quite serious i f a warm period follows the f i r s t frost* Good evidence exists that early frosts are responsible for low sugar content i s given by the fact that i n 1949 i n Alberta, a k i l l i n g frost occurred on September 12 and the average percent of sugar on September 30 was only 14*5 percent* It i s believed that this early frost was responsible for the abnormally low average sugar percent of 14*5 at the 30 day point* These results are based on a 20 years average* S t a t i s t i c a l methods have shown a positive correlation between frost and average sugar percent of harvested beet roots (1)* Late second growth of tops causes a reduction i n sugar content* Where early frosts are followed by warmer periods, second growth i s favoured* sucrose loss maynot occur when second growth i s absent and the i n i t i a l frost period persists, keeping the a i r and s o i l temperature low (2)* Storing sugar beets i s a common practise and the question of loss of sugar and weight during storage has re-3. ceived careful attention, sugar loss results from respiration occurring i n the root tissue* sugar beets w i l l not die during storage unless badly frozen while being stored* prom experiments keeping storage beets at low temperatures, there i s evidence that sucrose percent does not decrease as long as the beet remains close to freezing* This means a temperature of -1*7°C (2)* Respiration i s accompanied by a change of the sucrose into the invert sugars glucose and fructose* The sucrose loss i s greater i n beets which have not been decrowned* Under favourable conditions of temperature "sprouting" occurs* This w i l l cause a sucrose reduction* Temperature i s important in the degree of loss, that i s , the higher the temperature the more active are the changes* I f a suitable temperature for sugar conversion and respiration be accompanied by drought, then the loss i s high (2)* Thus, leaf regrowth during pile storage and the resulting sucrose reduction may be a problem which i s similarly, If not directly, related to sucrose loss and leaf regrowth following autumn frosts* The problem of sucrose disappearance following frosts i s one of economic importance since the loss of sugar i n the t beet root may be aa highaas l . l percent ten days after frost (1)* The concern of the plant biochemist i s i n how these frosts i n i t i a t e metabolic changes i n the root which result i n the loss of sucrose and also, what metabolic changes are i n -volved, i t i s the purpose of this experiment to discover some of tho metabolic changes occurring in the beet root following f o l i a r loss and subsequent leaf regrowth. The sugar beet industry in Canada has shown steady growth since 1881. The most recent figures that could be obtained for the Canadian sugar industry show that i n 1952 the total yield of beets was 1,022,693 tons, harvested from 92,607 acres and valued at 12,681,393 dollars (3). i n 1950, the three Alberta factories located in the south of the province at Raymond, picture Butte and Taber, processed 349,000 tons of beets from a harvested area of over 32,000 acres. On the basis of the l a s t mentioned beet tonnage, a loss of 1.0 per-cent sucrose would be equivalent to 700,000 pounds or 350 tons of manufactured beet sugar. Studies of the specifio reasons for sucrose loss i n beet roots which have had their foliage removed are few. L i t t l e effort i n the past has been given to the biochemical aspects of the problem, some related physiological and bio-chemical aspects are outlined i n the following literature review: A study of twenty years harvest results and average sugar content for the Canadian sugar Factories at Raymond, Alberta, have shown, that k i l l i n g frosts of 26°p. prior to harvesting almost invariably cause a drop i n sugar i n the root and that sugar losses are most severe when rapid regrowth of top8 occurs after freezing (4). 5. prom experiments conducted on the defoliation of sugar beets by F* strohmer et a l (5)., the conclusion i s t h i t complete as well as partial defoliation of the sugar beet reduces both the total yield of sucrose and the total weight of the beets* A relationship between the, number of leaves and the accumulation of dry mass and sugar i n beetaroots has been noted (6). Removal of more than half the leaves (2-4 l e f t ) caused a pronounced decline i n the yiel d of dry matter of the root but only a slight decrease in the percent sugar per plant* Increase i n productivity of the remaining leaves i s perhaps due to the more rapid removal of free carbohydrate from the green tissues* When there.are many leaves, the excess of photosynthate might remain where synthesized and hinder the assimilating process in the root* Enzyme studies using beet root tissue have shown the following: A*I* Oparln (7), i n his studies of beet root invert-ase, succeeded in establishing the presence of the enzyme i n sugar beet root* It was found possible to reveal hydrolytic activity i n suspensions prepared by maceration of the top parts of the root, but i n the central portion of the root, i n -vertase activity was apparently absent* The following relationships between invertase activity and sugars fere found: 6. 1. Tne variety with the maximum Invertase activity has the lowest total sugars and percent dry material. 2. An invertase activity increase i s accompanied by lower proportional sucrose and higher proportional mono-saccharide i n beet roots. 3. An inverse relationship exists between invertase activity and the ratio of sucrose to monosaccharide. Oparin believes that invertase activity i n beet roots i s mainly synthetic and that glucose and fructose translocated from the leaves forme i sucrose i n the root as a result of i n -vertase synthetic action. The exact role of Invertase i n sugar beet root i s unknown. J. Tyson (8) found that catalase activity i n beet leaves i s positively correlated with vigor of growth and size of sugar beet plants. Catalases are unevenly distributed i n the beet root, being more aetive near the epidermis and de-creasing towards the centre of the beet (9). E. Ffankuoh (10) proposes that the function of sugar beet phosphatase i s to control carbohydrate metabolism i n which phosphorylation has an important part, i t i s suggested that phosphatase serves as a "brake11 on the degradation of carbohydrate by sp l i t t i n g off the phosphate from either the glucose or fructose monophosphate esters, thus preventing them from being oxidized in* the Embden Meyerhof pathway. Beta Amylase and starch phosphorylase enzymes are active i n beet root tissue and can be estimated from tissue preparations* There have been various studies by other workers of the sugars present i n normal beet roots* These studies have concerned synthesis, translocation, and relation of the sugars to other beet root constituents. The common sugars found i n the beet root are sucrose, raffinose, glucose, fructose, and arabinose. sucrose comprises on the average 65.0 percent of the dry weight of the beet root; raffinose about 0.6 to 1.0 percent; glucose and fructose 1.0 to 2.0 percent; arabinose i n traces (2). prom studies of the presence of reducing sugars i n freshly harvested beets by H. pellet (11), the conclusions made were: 1. Freshly cut beets contain from 0*04 to 0*10 per-cent reducing sugars based on fresh weight and most contain from 0.08 to 0.05 percent* 2. There i s no relation between the percent of sucrose and the percent of reducing sugars. 3. The amount of reducing sugars i s practically constant throughout growth. From researoh of other workers (12) interested i n sugar beet carbohydrates and their metabolism, i t has been 8* noted that: 1. The percentage of fructose Increases as the grow-ing season progresses. A low level of fructose i s associated with rapid growth and a higher level with slow growth and maturity. 2. Fructose, glucose, and sucrose a l l show diurnal variations within the leaf blade. Variations i n fructose and glucose together are usually twice that of sucrose. Dextrins are an important secondary product of photosynthesis i n the beet. 3. sugars move out of the blade mesophyll into the veins i n a polar direction. 4. The proportion of reducing sugars Immediately above the crown of the root i s 50 percent of the total sugars, while the crown contains only 1.0 percent reducing sugars. This indicates sucrose synthesis i n the crown of the beet. J. peklo (13) found that the condensation of soluble carbohydrates to starch in the cells of the sugar beet root is connected with the presence of leucoplasts in the c e l l s . Starch synthesis can readily he noticed i n roots with a high percent of sugar. The relation of nitrogen to accompanying changes i n composition of beet roots has been followed (14). The ut i l i z a t i o n of sucrose reserves has been found to depend upon the supply of nitrogen available to the plant. 9* Studies of the metabolism in sugar beet root at low temperatures (15) have shown that below -2°c, respiration with loss of sucrose continues slowly u n t i l freezing i s complete. One Kilogram of beet root tissue at -12°c evolved 1.6 milligrams of C O 2 in 1 hour. Respiration at -12°C con-tinued 4 days. On thawing, i t was found that sucrose i s con-verted into glucose and fructose. Through complete freezing by severe natural frost, loss of sucrose i n the beet root i s reduoed more than 35 percent i n relation to sucrose loss caused by partial freezing. The most recent research on the problem of sucrose synthesis i n higher plants has been done by D.P. Burma and D.C. Mortimer (16). These investlgatirs found that when radio-active glucose -1 -phosphate (G-l-P) i s fed to an excised sugar beet leaf through i t s petiole, radioactive sucrose, equally labelled i n the two moieties and a trace of uridine diphosphate gluoose (TJDPG) can be detected. Radioactive G-l-P i n the presence of uridine triphosphate (UTP) or a mixture of uridine diphosphate (UDP) and adenosine triphosphate (ATP) yields radioactive TJDPG i n the leaf homogenates. The enzymatic activity i s associated with the c e l l sap cytoplasm. G-l-P can not be replaced by free glucose, fructose or sucrose for the biosynthesis of TJDPG. On adding a mixture of radioactive G-l-P, UTP and fruotose-6-phosphate (F-6-P) to the homogenates, radioactive sucrose i s formed within f i f t e e n minutes. The mixture of G-l-P and UTP can be replaced by TJDPG but p-6-p 10. cannot be replaced by either free fructose or fructose 1-6 diphosphate for the biosynthesis of sucrose, i t i s possible that the Intermediates i n sucrose synthesis i n the beet leaf are afctive i n beet root -tissue. Such substances as oxalate, asparagine, glutamine, allantoin and betain can be detected in considerable concentration i n sugar beet roots. 2. THE SUGAR BEET PLANT The sugar beet belongs to the goosefoot family, or Ghenopodiaceae. The chief cultivated members of this family are beets and spinach. The species Beta vulgaris includes sugar beets, mangel wurzels, common garden beets, and lea f beets. There i s a wild form of the same genus (Beta maritima) which grows as a perennial along the coast of northern Europe* The sugar beet i s ordinarily a biennial, storing sucrose i n the root during the f i r s t year, and sending up seed stalks i n the second. They bear the flowers, seeds, and most of the leaves. The stem, i n the f i r s t year, consists of the crown on top of the root. The crown i s very much short-ened and scarcely distinguishable from the fleshy root. A The enlarged fleshy taproot i s the predominate part of the beet plant. A cross or transverse section of the root w i l l show a number of concentric rings, and between each there i s a soft, thin-walled cellular material or storage 11. parenchymatous tissue containing the c e l l sap. The coneentric rings are made up of collateral fibro-vascular bundles comprising the phloem, the cambium, and the xylem. The phloem i s composed of sieve tubes, companion cel l s , and thin-walled meristematic cells of rectangular shape, which are joined with similar cells arising i n the medullary rays, thus giving the appearance in mature roots of complete rings. These are known as cambium rings and add new c e l l s to the phloem and xylem. The xylem i s composed of vessels (tracheae) and woody parenchyma having thickened c e l l walls. These vessels contain protoplasm i n their early stages of develop-ment, but this disappears and secondary thickenings appear on the c e l l walls. The vessels serve i n water and dissolved mineral transport (17). The formation of secondary cambial zones i s responsible for many of the unusual types of stems encountered. In the beet family there i s f i r s t formed a hollow cylinder of irregularly arranged bundles. These bundles are partly of a secondary nature, but camblal activity soon ceases and a new, secondary cambium arises i n the pericycle just outside the bundles. The activity of the secondary cambium layers i n the beet root i s more nearly l i k e that of usual cambium but i s complicated by prolonged primary growth. The beet "root" consists of root, transition region, and several internodes of stem. The f i r s t cambium forms a ring of bundles close about the primary xylem. A secondary cambium arises i n the 12. pericycle and this i s followed i n rapid succession by others originating similarly. A l l layers continue to function, perhaps indefinitely* though more slowly after an early period of activity* The cambium arises apparently as a continous band, but forms more or less separate bundles with bands of conjunctive parenchyma developing between the vascular strips* The position of each new cambium, as i t arises i n the pericycle, is such that i t encloses a few layers of pericyelic c e l l s * These multiply and build a parenchymatous layer as rapidly as the cambium builds the vascular layer* Alternate bands of proliferated pericycle and of vascular bundles are then foisned. The former constitute the dark-colored, the lat t e r the l i g h t -colored rings in the beet root* The bundles are largely parenchymatous with only a few l i g n i f i e d cells i n the xylem. Growth continues In the bundles apparently both by oambial activity and by proliferation of the parenchyma of the xylem and the phloem* In this way the beet increases i n diameter by growth throughout i t s layers* The layers are not always complete cylinders and are united Irregularly with other layers, so that a complex, asymmetrical structure i s formed(18)* The sugar beet produces perfect flowers which are i n small dense cymes. The perianth i s uniseriate, consisting of five connate sepals* Petals are absent* There are five stamens opposite the calyx lobes and partly attached to the perianth ring* The filaments are distinct and the anthers 2-celled, dehiscing longitudinally. There Is one p i s t i l and 13. the inferior ovary i s pa r t i a l l y embedded in the flesh of the receptacle. The ovary i s 1-loculed, 1-ovuled, 3-carpelled. The f r u i t i s an indehlscent nutlet. The flowers are produced in dense cymes along an axis, resulting i n the formation of multi-seeded "seed b a l l s " . In germination, the primary root f i r s t appears followed by the cotyledons (epigeal germination). The seedling consists of a short hypocotyl, two fleshy cotyledons, and a primary root from which a few fibrous laterals arise (19). 13. MATERIALS AMD METHODS -CULTIVATION OF THE SUGAR BEETS FOR THE EXPERIMENT S.K.E-R-ll sugar beet seed~obtained from the B.C. Sugar Co. L t d . , Vancouver, B.C., was germinated i n f l a t s on January 2 9 , 1 9 5 4 * The seedlings were thinned and transplanted on March 25 to other f l a t s . Approximately s i x t y sugar beet seedlings were placed i n each f l a t : and 15 f l a t s prepared. The plants were kept under moderate l i g h t conditions i n a greenhouse from where they were transplanted to the f i e l d on May 2 , 1954. The beet roots were, on the average, 3 inches i n length. The young plants were spaced 8 to 12 inches i n the f i e l d and the rows planted l£ f e e t apart. Approximately 900 beets were planted consisting of 15 rows with 60 beets per row. The s o i l condition at the time of planting was not favourable f o r beet roots. I t was mainly a sandy loam which had become packed at the 6 inch below surface l e v e l . The f e r t i l i z e r content of the s o i l was considered as being^adequate f o r proper beet root growth and commercial f e r t i l i z e r s added r M^&^-srplanting. The beetB were well watered during the summer months by a s p r i n k l e r . The p r e c i p i t a t i o n during the period from May to August, 1954» was quite heavy and many days were overcast and d u l l . ThiB climatic condition as well as the s o i l condition would tend to favour a low sugar content. The beets, at harvest time i n August 1954» had not reached commercial maturity and were continuing to store sugar i n the root. The average root s i z e was 3<*4 inches across the centre of.the root and approximately 6 inches i n length from crown to tip . METHOD USED TO RANDOMIZE THE BEST SAMPLES AND FREEZE THE ' BEET LEAVES Seven rows of beets with the best f o l i a r appearanoe were selected. Lots of ten beete were chosen from each row, giving a total number of 30 lots amongst the 7 rows. Each 8 i n g l e l o t was given a number and then the numbers were ran-domly picked. Diagram II shows the arrangement of the plot. This randomized l o t design was chosen i n order to reduce errors caused by s o i l differences, moisture variations, pH, and s o i l structure etc. Since the total area of the beet plot was small, differences of temperature, wind, and l i g h t were not considered. Of the 30 groups of 10 beets, 6 randomly selected groups had their leaves frozen, and other such selected groups were defoliated, decrowned, continously defoliated, or used as controls. The method of freezing consisted of covering 6 beets at a time i n the f i e l d with an insulated box approximately 48 X 16 X 24 inohes (see diagram I I I ) . A wire mesh tray con-talning about 40 pounds of dry (CO2) ice was placed in a rack in the upper portion of the box. A 60 minute treatment period with such an apparatus was used to destroy the tops without noticeable damage to the crowns. Temperature measure-ROWS 5 4 4 10 9 8 I 14 13 18 17 12 16 22 21 i i 20 26 t 25 24 i i 29 I 28 6 11 1 5 19 S 23 27 DIAGRAM II - BEET PLOT ARRANGEMENT KEY -FROZEN DEFOLIATED GONT. DEFOL. - CROWN CONTROL 3 4 5 2 1 14 8 7 9 10 12 15 16 13 11 21 22 29 17 18 23 24 25 * 9 26 30 27 20 28 6 i£. . merits made during the freezing period gave readings of -16° C. at 2 inches above ground level ami-Ae,'. the box, and an average air temperature within the box of -11° C , 2 inches below the dry ice. The outside air temperature was 16° C. At the end of 1 hour, the leaves and petioles but not the beet root were completely frozen* Some beet crowns were pa r t i a l l y exposed to freezing while others were beneath the s o i l surface. No attempt was made to protect the crown as i t is usually exposed in f i e l d beets grown commercially* Also, i t was desired to duplicate fi.«)Wx conditions as closely as possible. The weather was humid and overcast on August 16, 1954, the day of freezing* One hundred and twenty of the beets were defoliated with a knife by sli c i n g off the leaves one-quarter inch above the crown* A l l dead leaves s t i l l attaohed to the crown were also removed. Sixty of these beets were not treated following defoliation* The remaining defoliated beets were periodically redefoliated every two days. Sixty beets were decrowned. Decrowning was accomplished by cutting the beet root transversely just be-neath the outer ring of meristematic buds. The out surfaces were l i b e r a l l y coated with paraffin wax, this being done to prevent fungous and bacterial attack. The defoliated beets were used to serve as a parallel to the destruction of leaves by freezing. The continually DIAGRAM III - GUT-AWAY END VIEW OP BOX USED TO FREEZE BEETS IN THE FIELD d e f o l i a t e d beets served as a check on the d e f o l i a t e d beets where i t was c o n s i d e r e d t h a t p h o t o s y n t h e s i s i n the new r e -growth l e a v e s would p a r t i a l l y o f f B e t a l a r g e sugar l o s s i n the r o o t * No photosynthate would be t r a n s l o c a t e d to the beet r o o t as l o n g as the l e a v e s were removed, hence d i f f e r e n t carbohydrate changes were expected i n the r o o t s of the con-t i n u a l l y d e f o l i a t e d b e e t s . Beets w i t h t h e i r crowns removed were used i n o r d e r to determine whether sugar changes would occur as r a p i d l y when the m e r i s t e m a t i c c e n t r e (crown) had been d e s t r o y e d . The f i r s t experiment and treatment began on August l 6 , 1954«, three and one h a l f months f o l l o w i n g t r a n s p l a n t i n g to the f i e l d . At v a r i o u s time i n t e r v a l s from treatment, the beet s were h a r v e s t e d i n groups of ten, A group c o n s i s t e d of 10 beets a l l t r e a t e d i n one s p e c i f i c manner. Groups were h a r v e s t e d i n the f i r s t experiment a t the 1, 4, 8, 11, 16, and 20 day i n t e r v a l s f o l l o w i n g August 16. The second experiment and treatment commenced on October 13, 1954,, w i t h h a r v e s t s on the 1, 4, 8, 12, and 15th days f o l l o w i n g treatments. Enzymes o n l y were determined i n t h i s experiment. The b.e.et r^tqpips used were s i m i l a r as i n the f i r s t experiment* The topB of the d e f o l i a t e d and c o n t r o l beets were weighed when removed(at the b e g i n n i n g of the experiment f o r the <UfeJ»*tij4 b e e t s , and a t each h a r v e s t f o r the c o n t r o l s . The f r e B h l e a f weights of the d e f o l i a t e d and c o n t r o l beets were recor d e d and l a t e r compared w i t h l e a f regrowth weightB and sugar c o n t e n t . The average f r e s h weight of the regrowth l e a v e s was o b t a i n e d f o r the d e f o l i a t e d beets when they, were topped a t each h a r v e s t p e r i o d . L e a f regrowth weights c o u l d o b v i o u s l y not be measured i n the case of the c o n t r o l groups. PROCEDURE USED IN THE PREPARATION OP BEET SAMPLES The beet r o o t s used i n t h i s experiment were o f the "fangy" type. They were g l o b u l a r i n shape and not the t y p i c a l cone. Sugar grooves were absent and the s i n g l e and even t r i p l e primary r o o t t i p s were t i g h t l y t w i s t e d and c u r l e d about the bottom of the b e e t . Pew of the r o o t s o b s e r v e d p e n e t r a t e d d i r e c t l y downwards i n t o the s o i l . T h i s growth-l i m i t i n g c o n d i t i o n was caused by the compact s t r u c t u r e of the s o i l . Secondary r o o t b r a n c h i n g was a l s o i n h i b i t e d . The procedure was as f o l l o w s : 1. The 10 beets of each group were p u l l e d from the s o i l l e a v i n g behind most of the secondary r o o t s . The new l e a v e s were s l i c e d o f f the f r o z e n and d e f o l i a t e d b e e t s . The l e a v e s were a l s o removed from the c o n t r o l b e e t s . Transverse k n i f e s l i c e s were made approximately i n c h below the c e n t r e of the crown r e g i o n where new l e a v e s appear. Any s e v e r e l y t w i s t e d r o o t s were removed. 2. Each beet was s l i c e d d i a g o n a l l y across i t s c e n t r e r e g i o n . ( s e e diagram I o f beet t i s s u e r e g i o n s and sugar com-p o s i t i o n ) . T h i s was done i n order to a c q u i r e t h a t p o r t i o n of P l f t f e f t f l M I - S H O W I N G SOC ROSE: O l S r R I B o r j O N I N piFPe^erMr «E & I O N « S T H E S U & - A R Beer R O O T - -_ A N ^ Y S ' ^ 6 V H . 8 > P U £ tA - 6 E E . T CrROvJER'S A N N U A L l<\0&. the beet where sugar concentration i s highest* The size of the sections was approximately 2 inches in thickness and 4 inches i n length* 3* The sections were put into a meat grinder and pulped. This pulp was then well mixed for 10 minutes, i n -eluding with i t the small amount of beet juice produced by t pulping* About 2000 grams of pulp was obtained from 10 beet sections* 4* Approximately 300 grams of fresh pulp was weighed, placed in the 110° C. oven for 10 minutes, removed and trans-ferred to the 65° 0. oven where i t was dried to constant weight (4 days). The high temperature for 10 minutes i n -activates a l l enzymes and low temperature drying avoids cara-mel ization of the sugarB. The material was ground to 40 mesh in an electric pulverizer and stored over calcium chloride i n dessicators. . 5. Forty grams of fresh pulp from each group was weighed to the nearest tenth of a gram and blended in a Waring blendor with 100 mis. of d i s t i l l e d ice water for 2 minutes. The solution was f i l t e r e d through broadcloth and the f i l t e r e d extract kept cold and used immediately i n enzyme activ i t y estimations. 6. The remaining pulp was heated to 110° C. for 10 minutes, transferred to cpl^s+tfc-aie bags and stored in a -10° C refrigerator for future analyses. 1 *. The beet roots harvested in the second experiment in October were treated in the same manner and used for enzyme I - ReFRItERflTAON O B T A I N E D T H f o u t H THfc K I N D N E S S o.F. DR. W o o O •. PBPT- O F A N I M A L rtuSBANORV. vi. 3 . C . rune. Forty seven grams of pulp was "blended with 100 mis, of d i s t i l l e d i c e water f o r two minutes. Diagonal sections of the beet root were pulped f o r the beta-amylase and phosphorylase determinations while •§• inch of the crown portion was used i n the estimation of invertase a c t i v i t y . An experiment to deter-mine the region of beet root having the greatest invertase a c t i v i t y was carr i e d out. The r e s u l t s are given i n Table I . VALIDITY OF THE SAMPLING METHOD On consideration of the sampling technique used i n this experiment, i t was thought that a check on the method would show i f 40-gram portions of beet pulp blended f o r enzyme a c t i v i t y estimations a c t u a l l y represented the sample. The test f o r v a l i d i t y was accomplished by harvesting f i v e average s i z e beets, cleaning, s l i c i n g and pulping them i n a meat grinder. The t o t a l volume of pulp was thoroughly mixed for 10 minutes, and 5* 40-gram aliquots weighed out. These pulp aliquots were blended with 100 mis. of d i s t i l l e d i c e water as quickly as possible and used immediately f o r the es-timation of phosphatase a c t i v i t y . Sucrose analyses on the fresh, pulp were also made. The r e s u l t s are given i n Table I I . SUBSTANCES ANALYZED FOR; Analyses were carr i e d out f o r the following: Fresh pulp Dried, ground pulp Alcohol ex-tracted pulp Catalase Total nitrogen Phosphatase Sucrose Insoluble nitrogen 8,1. Fresh pulp Phosphorylase Invertase beta-amylase Dry weight sucrose invert sugars These analyses should indicate any major changes occurring i n the metabolism of the beet root* The enzymes selected were those which could be determined with the equip-ment available in the laboratory. A Warburg respirometer for respiratory enzyme activity measurement was not standard equipment i n the laboratory at the time of this experiment* Catalase activity i s indicative of general c e l l metabolism and plant growth (8). phosphatase i s Important i n the glycolytic scheme and the control of carbohydrate meta-bolism i n sugar beets (10)* phosphorylase i s active i n starch metabolism: Glucose-1-phosphate starch 4 phosphate phosphorylase Beta-amylase functions i n the breakdown of starch to maltose and high molecular weight dextrins. Beta-amylase possibly has a role i n the carbohydrate metabolism of sugar beet root. Invertase activity has been correlated with sucrose content of beet roots by some investigators (7) . pried, ground pulp Alcohol extracted pulp invert sugars starch-dextrins TABLE I INVERTASE ACTIVITY IN VARIOUS REGIONS OP THE BEET ROOT Section of beet Invertase activity expressed as mgms of invert sugar X 1000 Grown 300 Beet centre 290 Outer surface 270 Beet tip 210 TABLE II VALIDITY OF THE SAMPLING METHOD Pulp Aliquot phosphatase percent sucrose activity as fresh weight ugms of phenol I 492 15.1 II 487 15.0 III 501 15.3 IV 496 15.0 V 485 14.9 zz. I n a l l analyses, d u p l i c a t e determinations were made of each sample., Percentages are based on both dry and f r e s h weights. The value T/C i s obtained by d i v i d i n g the value f o r substance or a c t i v i t y i n the treated p l a n t s by the value ob-tained f o r that substance or a c t i v i t y i n the c o n t r o l p l a n t s and m u l t i p l y i n g by 100. In the t a b l e s , the value i s taken to the u n i t p l a c e . 14'. ANALYTICAL METHODS - FRESH PULP PHOSPHORYLASE - The a c t i v i t y of t h i s enzyme was measured by the method of Sumner e t a l , (26). One ml. of en-zyme e x t r a c t i s incubated w i t h 2 mis. of b u f f e r e d s u b s t r a t e -s t a r c h substrate f o r 1 hour a t 25° 0 . The ^ . t y l f e ^ d L s u b s t r a t e c o n s i s t s of 1 gram of glucose-l-phosphate i n 50 mis. of d i s t -i l l e d water. Shaking w i t h dry calcium oxide removes i n o r g a n i c phosphates. The f i l t r a t e from t h i s i s n e u t r a l i z e d w i t h drops of HOI and an equal volume of pH 6 . 0 c i t r a t e b u f f e r added. This substrate i s mixed with an eqmal volume of a 1 percent potato s t a r c h s o l u t i o n before use i n a determination. The b u f f e r e d substrate w i l l keep a t room temperature w i t h a thymol c r y s t a l f o r l-jt weeks. D i g e s t i o n i s stopped by the a d d i t i o n of 5 mis. of 6.66 percent ammonium molybdate. Color development i s obtained by adding 5 ml. of 7 *5^ s u l f u r i c a c i d , f o l l o w e d by 5 ml. of 4 percent f e r r o u s s u l f a t e s o l u t i o n and then 10 m l . of d i s t i l l e d water. The mix i s r o t a t e d and allowed to stand ENZYMES f o r 10 minutes before a c o l o r i m e t e r r e a d i n g . A blank i s run by adding the su b s t r a t e a f t e r the a d d i t i o n of ammonium molyb-date to the enzyme e x t r a c t . Headings were made on a K l e t t c o l o r i m e t e r zeroed to a tube of d i s t i l l e d water and u s i n g a green f i l t e r . A c t i v i t y of phosphorylase i s expressed as micro-grams of phosphorus formed per m l . of e x t r a c t . A standard phosphorus curve was made by pre p a r i n g a s e r i e s of phosphorus s o l u t i o n s (as KH2PO4) w i t h a range of concentrations between 4 and 36O micrograms phosphorus. Color development and d i l u t i o n were c a r r i e d out as f o r an a c t u a l phosphorylase determination. PHOSPHATASE2- Phosphatase a c t i v i t y was determined by Gottschalk's micro method (2f<) , where a c t i v i t y i s measured as micrograms phenol formed per ml. of p l a n t e x t r a c t . The method c o n s i s t s of in c u b a t i n g 1 m l . of the p l a n t (enzyme) e x t r a c t w i t h 5 m l . of a phenyl-dieodium phosphate s o l u t i o n s u b s t r a t e f o r 1 hour a t 37° C. The su b s t r a t e c o n s i s t s of 1.09 grams of phenyl disodium phosphate i n 200 m l . of pH 6.0 c i t r a t e b u f f e r made to 1 l i t r e w i t h d i s t i l l e d water. The amount of l i b e r a t e d phenol i s estimated by c o l o r development u s i n g 2 ml. o f F o l i n -C i o c a l t e u reagent, which i s e s s e n t i a l l y unreduced phospho-molybdic a c i d . F i v e m l . of a 10 percent sodium carbonate s o l u t i o n ci'Sv added, and the phosphomolybdic a c i d , i n a l k a l i n e s o l u t i o n , i s reduced by phenol to a mixture of the lower oxides of molybdenum, and t h i s mixture y i e l d s the t u r b i d blue known as molybdenum b l u e . Affter 10 minutes, the mix i s d i l u t e d by adding 100 ml. of tap water. A blank i s run by adding 1 ml. of the enzyme e x t r a c t to a f l a s k i n which no e x t r a c t was added a t f i r s t . Color...intensity readings were made on a K l e t t photo e l e c t r i c c o l o r i m e t e r u s i n g a green f i l t e r . The i n t e n s i t y i s read a t a d e f i n i t e time which was 10 minutes a f t e r d i l u t i o n . A s e r i e s o f phenol s o l u t i o n s was prepared and used i n the c o n s t r u c t i o n of a standar d curve. The phenol s t o c k s o l u t i o n c o n t a i n e d 0.9 mgms. of phenol per m l . This was d i l u t e d 10 times, and 2 m l . to 10 m l . volumes o f the d i l u t e d s t o c k s o l u t i o n were analysed f o r ph e n o l . C o l o r development and d i l u t i o n were c a r r i e d o ut as f o r an a c t u a l phosphatase d e t e r -m i n a t i o n . CATALASE - C a t a l a s e a c t i v i t y was estimated by Sumner and Somer's m o d i f i c a t i o n o f the procedure of Von E u l e r , u s i n g 3» 6, and 9 minute i n t e r v a l s . (£2-). Two m i s . of the p l a n t e x t r a c t i s added to 2$ mis. of i c e c o l d c a t a l a s e s u b s t r a t e a t 0 time when 5 m l . of the mix i s withdrawn q u i c k l y and blown i n t o 5 ml of 2N s u l f u r i c a c i d , thus s t o p p i n g the r e a c t i o n . At the 3, 6, and 9 minute i n t e r v a l s a f t e r the withdrawal o f the f i r s t enzyme-substrate a l i q u o t , other J> ml. a l i q u o t e o f the mix are withdrawn and blown immediately i n t o 2N s u l f u r i c a c i d . The decomposition of hydrogen per o x i d e i s a r r e s t e d w i t h the a d d i t i o n o f s u l f u r i c a c i d . Ten m l . of a 5 p e r c e n t KI s o l u t i o n cvs. added to the e n z y m e - s u b s t r a t e - s u l f u r i c a c i d mix, and then a drop of 1 p e r c e n t ammonium molybdate. A f t e r 3 minutes, the l i b e r a t e d i o d i n e i s t i t r a t e d w i t h 0.00?N sodium t h i o s u l f a t e , u s i n g s t a r c h s o l u t i o n as an i n d i c a t o r . v The v e l o c i t y c onstants f o r c a t a l a s e a c t i v i t y were c a l c u l a t e d u s i n g the equation K 5 l / T l o g A/A-x. The velocity constant i s K, the minutes of digeXstion are T; A i s the titration value of 5 ml. of the digest at 0 time in terms of 0 . 0 0 5 ^ thiosulfate; A-x i s the titration value after T minutes. The various values obtained for K were plotted against time and the graph was extrapolated to estimate the value of K at 0 time. Catalase activity is given in the table as the monomolecular value extrapolated to 0 time. Several modifications itf the method of Sumner and Somerfe.: were used in this experiment. The f i r s t modification was that 2 ml. of extract and 25 ml. of substrate were used in-stead of the accepted 1 ml. of extract and 50 ml. of substrate. The second modification was made in preparing the substrate. The substrate employed i n these determinations contained 23.2 ml. of pH 6 . 8 phosphate buffer and 13.6 ml. of 3 percent hydrogen peroxide made up to 250 ml. with d i s t i l l e d water. The buffered mix aB prepared in Sumner and Somer's procedure contains 6 . 7 ml. of phosphate buffer pH 6 . 8 , and 11.3 ml. of 3 percent hydrogen peroxide and d i s t i l l e d water to make 1000' ml. These changes employed were the result of experiment-ation using various concentrations of 3 percent hydrogen per-oxide from 2.0 to 13.6 ml. in 250 ml. of d i s t i l l e d water. Two and 5 ml. volumes of plant extract were incubated with the different substrates and the reaction mix with the highest activity used i n this experiment. Catalase activity i n the beet root i s d i f f i c u l t to determine as a consequence of i t s c o n c e n t r a t i o n i n beet root t i s s u e * BETA-AMYLASE - ©eta Amylase a c t i v i t y was measured by Sumner and Somer's m o d i f i c a t i o n of the procedure of will-s t a t t e r and co-workers. (£3). I t c o n s i s t s of i n c u b a t i n g p l a n t e x t r a c t w i t h a b u f f e r e d s t a r c h s u b s t r a t e and measuring i o d i -m e t r i c a l l y the q u a n t i t y of maltose formed. Twenty f i v e mis* of a 1 percent s t a r c h substrate i s run from a b u r e t t e i n t o a glass-stoppered b o t t l e . Ten m l t . of a 0.2 M acetate b u f f e r Is added. The pH should be 4*4-4.5* At 0 time, 1 ml. of p l a n t e x t r a c t I s blown i n t o the s u b s t r a t e , which i s mixed and placed, i n a 37°C. incubator f o r 20 minutes-. At the end of t h i s p e r i o d of i n c u b a t i o n , 2 ml. of N HOl\e*sa. blown i n and the s o l u t i o n mixed. S i x t y ml. of O.lN. WaDH faise added and the s o l u t i o n again r o t a t e d to mix. Twenty ml. of 0.01 N IKI i s p i p e t t e d i n t o the b o t t l e . A f t e r 10 minutes, 10 ml. of 10 percent s u l f u r i c a c i d i s added and the mix r o t a t e d . T i t r a t -i o n i s done w i t h 0.01 N sodium t h i o s u l f a t e . A blank I s run by adding 1 ml. of the enzyme a f t e r the a d d i t i o n o f the s u l -f u r i c a c i d . The monomolecular r e a c t i o n constant f o r beta-^ m y l a s e was obtained from the equation: K s 1/t l o g ' 0.1875 • 0.1875 - x The time i n minutes i s represented by t , and x i s the c o n -nected t i t r a t i o n value. &3. INVERTASE - The method f o r the determination of i n -vertase a c t i v i t y i n p l a n t e x t r a c t s as o u t l i n e d by J.B. Sumner and S.P. Howell i n tho Journal of B i o l , ohom, (&:4), was found to be inadequate f o r the determination o f i n v e r t a s e i n sugar beet root e x t r a c t s . This method i n v o l v e s the use o f a sucrose b u f f e r e d substrain and the e s t i m a t i o n o f reducing sugars pre-sent before and a f t e r i n v e r t a s e a c t i v i t y . Since the beet r o o t i s i n i t i a l l y h i g h i n sucrose, the need f o r a d d i t i o n a l sucrose substrate i s not apparent. The method used i n t h i s experiment i s a combination o f two separate methods. One o f these was k i n d l y s u p p l i e d by D. HLbbert, of the B r i t i s h sugar Corporation L t d , i n Peterbor-ough, England. The other was obtained from a t r a n s l a t i o n of Oparin«s s t u d i e s » } The method i n use at the c e n t r a l l a b o r a t o r y , B r i t i s h Sugar Corporation i s o u t l i n e d b r i e f l y as f o l l o w s : The roo t s are minced as r a p i d l y as p o s s i b l e and equal weights of mincinga and water are blended together w i t h excess c h l o r o -form f o r 5 minutes. The e x t r a c t i s f i l t e r e d through a f i n e c l o t h . Ten grams o f sucrose ai:<S3 weighed i n t o a t e s t tube and d i s s o l v e d i n 25 ml. of the f i l t e r e d beet e x t r a c t ; 10 ml. of acetate b u f f e r , pH 4.92 and an excess o f chloroform as»e added, and the tube shaken. A f t e r the chloroform l a y e r separates, 10 ml. of the s o l u t i o n ase p i p e t t e d i n t o a 50 ml. volumetric f l a s k , 10 drops o f b a s i c l e a d acetate (30°Be) added, and the mixture made up to the mark w i t h d i s t i l l e d - !• ' 2:8 » water. The c l a r i f i e d mixture i s f i l t e r e d and the p o l a r i m e t e r reading taken i n a 200 mm. tube. The remainder of the b u f f e r e d e x t r a c t i n the stoppered tube i s incubated f o r 48 hours at 37° G. and the f i n a l p o l a r i z a t i o n i s determined a t the end o f t h i s time. I f the decrease I n p o l a r i z a t i o n i n 48 hours i s P, then the enzyme a c t i v i t y (E.A.) i s expressed as: E.A. = 5P W Invertase a c t i v i t y thus expressed i s assumed to be 1000 times p o l a r i z a t i o n r e d u c t i o n brought about by 1 ml. of e x t r a c t i n 1 hour at 37° C» Oparln's method f o r the determination of i n v e r t a s e a c t i v i t y i s as f o l l o w s : F i f t y grams of pulp s.vse macerated i n 200 ml. o f d i s t i l l e d water. Two a l i q u o t samples of t h i s suspension are p i p e t t e d out; one of these i s i n a c t i v a t e d by adding calcium carbonate and b o i l i n g f o r f i v e minutes. The p a r a l l e l sample, not i n a c t i v a t e d , s e r v e s f o r the determination of i n v e r t a s e a c t i v i t y . To a l l samples, sucrose i s added to the extent of 20 percent o f the weight of pulp taken, t h i s being e s p e c i a l l y important, where the i n i t i a l sucrose i s low. The i n a c t i v a t e d and untreated samples are maintained a t 30° C« f o r 20 hours, c l a r i f i e d w i t h l e a d acetate, and the reducing sugars determined. The d i f f e r e n c e between the t r i a l and the c o n t r o l samples c h a r a c t e r i z e s the h y d r o l y t i c a c t i v i t y o f the i n v e r t a s e . The procedure f o r i n v e r t a s e - d e t e r m i n a t i o n used i n t h i s experiment i s as f o l l o w s * .The beets are cleaned and t h e i r crowns s l i c e d o f f approximately \ i n c h below the de-f o l i a t e d beet sur f a c e . The crown s l i c e s are minced i n a gr i n d e r and 40 grams of these-mincings a iss blended w i t h 100 mis. of d i s t i l l e d i c e water f o r 2 minutes.. The suspension i s f i l t e r e d through b r o a d c l o t h . One gram of pure sucrose I s placed i n each of two, 250 ml, erlenmeyer f l a s k s , and 25 ml, of f i l t e r e d beet r o o t e x t r a c t d e l i v e r e d to each f l a s k . The s o l u t i o n s are mixed u n t i l the sucrose i s d i s s o l v e d . Ten ml. of acetate b u f f e r pH 4.92 aiiffi added to the f l a s k s and to one f l a s k approximately 1 gram o f calcium carbonate. The calcium carbonate-beet j u i c e s o l u t i o n i s b o i l e d f o r 2 minutes, c a u t i o n taken to avoid burning the sugar. Ten ml. of chloroform tise added to each f l a s k and the f l a s k s shaken q u i c k l y , covers are p l a c e d over the s o l u t i o n s . a n d they are incubated at 37° C. f o r 48 hours. At the end o f 48 hours, the s o l u t i o n s are analyzed f o r i n v e r t sugars u s i n g the somogyi/shaffer-Hartmann method (2-5). Enzyme a c t i v i t y i s expressed as mgms. i n v e r t sugars formed times 1000. The blank i n v e r t sugar value i s subtracted from the a c t i v e run to o b t a i n the number of mgms. of i n v e r t sugars formed by i n v e r t a s e a c t i o n . DRY WEIGHT Three hundred grams o f f r e s h pulp was weighed r a p i d l y on a balance, placed In an oven at 110° C. f o r 10 minutes, and t r a n s f e r r e d to a 65° c. oven where i t was d r i e d to a constant weight. Drying u s u a l l y r e q u i r e d three or four days and i t was necessary to mix the pulp s e v e r a l times during the d r y i n g p e r i o d . Dry weight i s expressed on a per-centage b a s i s . SUCROSE Sucrose was determined by employing the method as o u t l i n e d i n the A s s o c i a t i o n of A g r i c u l t u r a l chemists (2/6). • I t i s the hot water method of d i g e s t i o n which proceeds as f o l l o w s : Twenty-six grams of f r e s h or frozen pulp (thawed before weighing) i s weighed a c c u r a t e l y on an a n a l y t i c a l b a l -ance and t r a n s f e r r e d w i t h water to a 201.2 cc. Kohlrausch f l a s k . The f l a s k i s h a l f f i l l e d w i t h c o l d , d i s t i l l e d water and placed i n a hot water bath a t 80° c. The f l a s k i s r o t a t e d at i n t e r v a l s and small amounts o f 80° 0. d i s t i l l e d water are added from time to time so tha t at the end o f 30 minutes the water i n the f l a s k i s a l i t t l e above the mark. At t h i s time, the f l a s k i s removed from the water bath and the s o l u t i o n allowed to c o o l to 25° C. S u f f i c i e n t concentrated a c e t i c to the ex+i-CLtt-a c i d i s added*to make very s l i g h t l y a c i d ( l e s s than 0.5 cc),alon<j awJSva. few drops o f ether. Ether i s added to reduce foaming, i n the neck of the Kohlrausch f l a s k . The volume i s com-p l e t e d to the mark. About 100 ml. o f the hot water e x t r a c t i s t r a n s f e r r e d to a metal cup or a f l a s k c o n t a i n i n g one-half gram of le a d acetate, shaken w e l l , and the s o l u t i o n allowed to stand undisturbed f o r a few minutes. I t i s then f i l t e r e d u s ing c e l i t e a n a l y t i c a l f i l t e r a i d , and the f i l t r a t e p o l a r -30. i z e d i n a 200 mm. tube. A Re i c h e r t p o l a r l m e t e r was used. Readings at room temperature are i n percent sucrose f r e s h weight, sucrose i s al s o expressed on a percent dry weight b a s i s , INVERT SUGARS Determinations were made on 5 ml, p o r t i o n s of the hot water e x t r a c t used i n the a n a l y s i s f o r sucrose, -The 5 ml, a l i q u o t used f o r i n v e r t sugar a n a l y s i s was p i p e t t e d d i r e c t l y from the Kohlrausch f l a s k and d i d not c o n t a i n l e a d acetate. The somogyi-Shaffer-Hartmann method f o r i n v e r t sugar a n a l y s i s was employed. (25). The procedure f o r t h i s method i s : P i p e t t e 5 ml,.of sugar s o l u t i o n c o n t a i n i n g 0,2 to 2,0 mgms, o f glucose i n t o a l a r g e t e s t tube and add e x a c t l y 5 ml. o f i n v e r t sugar reagent. Mix, stopper l o o s e l y w i t h a g l a s s stopper and place i n a b o i l i n g water bath f o r 15 minutes. Remove the tube, c o o l i n c o l d Water f o r 3 minutes u n t i l the temperature o f the s o l u t i o n i s 30-40° 0. Add 1 ml. of 5N s u l f u r i c a c i d , mix, and a l l o w to stand f o r 2 minutes. T i t r a t e w i t h f r e s h 0.005 N sodium t h i o s u l f a t e u n t i l most o f the Iodine has reacted. Add drops o f s t a r c h i n d i c a t o r and t i t r a t e to c o l o r l e s s end-point. For the blank, use 5 ml. o f d i s t i l l e d water i n place o f the sugar s o l u t i o n . Convert volume o f t h i o s u l f a t e i n t o mgms. i n -v e r t s by use of the s&S conversion t a b l e , i n v e r t sugars are expressed i n terms of both percent o f f r e s h and dry w e i g h t s ^ ) of pulp'. 32., IS. ANALYTICAL METHODS R' DRIED AND GROUND PULP  TOTAL NITROGEN One gram p o r t i o n s of the d r i e d and ground pulp were used to determine t o t a l n i t r o g e n by the standard K j e l d a h l method. This method, as des c r i b e d i n Loomis and S h u l l (211) r e q u i r e s d u p l i c a t e 1,000 gram samples of the o r i g i n a l d r i e d powder. The powder i s weighed on a 7 cm, f i l t e r paper. The paper i s f o l d e d and deposited i n a 800 ml, K j e l d a h l f l a s k , A s e l e n l z e d c r y s t a l I s added and 25 ml, o f a s u l f u r i c a c i d -s a l i c y l i c a c i d mixture poured i n . This a c i d mix i s prepared by adding 1 gram of s a l i c y l i c a c i d to 25 ml. of s u l f u r i c a c i d . The f l a s k i s r o t a t e d f r e q u e n t l y f o r 30 minutes. At t h i s time, 5 grams ~of sodium t h i o s u l f a t e are added and-the f l a s k warmed s l i g h t l y f o r 5 minutes, then cooled. E i g h t grams o f sodium s u l f a t e and a c r y s t a l o f copper s u l f a t e aise p l a c e d I n the f l a s k and the mix r o t a t e d . Heat i s a p p l i e d g e n t l y u n t i l f r o t h i n g i s over, and then s t r o n g l y u n t i l c l e a r . A f t e r 30 minutes, the f l a s k I s removed from the burner and cooled I n a fume c l o s e t , 250 ml. o f water ase added, the f l a s k I n c l i n e d , and 100 ml, o f 33 percent NaOH poured I n , The a c i d and base are not mixed, s e v e r a l pieces o f z i n c metal are added. The f l a s k I s then connected to the d i s t i l l a t i o n apparatus and ro t a t e d w e l l . The d i s t i l l a t e i s c a r r i e d - o v e r i n t o 50 ml, of 0,14 N HOl s o l u t i o n . Methyl red I s employed as an i n d i c a t o r . Approximately 150 ml. o f d i s t i l l a t e sts& c o l l e c t e d . The un-used a c i d i s t i t r a t e d w i t h 0.14 N NaOH. One ml. o f 0.14 N 33. NaOH i s e q u i v a l e n t to 1 mgm. o f n i t r o g e n . T o t a l p r o t e i n equals the number of mis. o f NaOH m u l t i p l i e d by 6.25. One gram o f sucrose i s used i n p l a c e o f the powder f o r the blank. T o t a l n i t r o g e n i s expressed on a dry weight b a s i s . is'. ANALYTICAL METHODS - ALCOHOL EXTRACT OP DRIED AND  GROUND PULP SOXHLET EXTRACTION E x t r a c t i o n o f the dry ground pulp w i t h elbahol was* done u s i n g the method o u t l i n e d i n Loomis and S h u l l (Z.3). i Two grams o f d r i e d powder at$3 weighed a c c u r a t e l y and placed i n an e x t r a c t i o n thimble. A loose c o t t o n p l u g I s I n -serted and the thimble deposited i n the s o x h l e t e x t r a c t i o n One Won <J red «*nd £%-H;j n\\ -column. i c O ^ r . I . o f 70 percent e&footaol atsa added and an ex-t r a c t i o n run f o r 45 minutes. The ethanol i s removed and 50 v ml. o f f r e s h 70 percent ethanol again added and an e x t r a c t i o n made f o r a f o l l o w i n g 2 hours. Before each ^ - t a ^ l change, the e x t r a c t i o n cup i s allowed to f i l l and the e&bohol r e -maining i n the f l a s k I s re p l a c e d . A second 50 ml. ethanol p o r t i o n ispused a t the end o f the 2 hour e x t r a c t i o n p e r i o d and the e x t r a c t i o n run overnight. The completion o f s o l u b l e sugar e x t r a c t i o n was determined by withdrawing 5 ml. of efchs-a h o l e x t r a c t surrounding the thimble, adding 2 drops o f a 15 percent a l c o h o l i c s o l u t i o n o f alpha naptho1 and 2 ml. o f concentrated s u l f u r i c a c i d . The presence o f a purple r i n g I n d i c a t e d incomplete carbohydrate e x t r a c t i o n . The e l f c a w i l i c 3*. e x t r a c t i s poured i n t o a 250 ml. f l a s k . The e x t r a c t i o n thimble i s heated i n a 70° G. oven u n t i l the e x t r a c t e d powder has d r i e d to constant weight. CLEARING AND DELEADING The a l c o h o l i c s o l u t i o n from the soxhlet e x t r a c t i o n was evaporated to 50 ml. and 50 ml. o f d i s t i l l e d water added. The s o l u t i o n was s t i r r e d u n t i l the r e s idue had broken up and d i s s o l v e d . This hot water e x t r a c t was t r a n s f e r r e d q u a n t i t -a t i v e l y to a 250 ml. volumetric f l a s k . A l l equipment was thoroughly r i n s e d f r e e from e x t r a c t w i t h d i s t i l l e d water. The e x t r a c t was cooled and 2 ml. o f a s a t u r a t e d , n e u t r a l l e a d acetate s o l u t i o n added. A f t e r making to volume, the e x t r a c t was- f i l t e r e d u s i n g s u c t i o n . Approximately 0.3 grams o f pot-as alum* oxalate were added to the f i l t r a t e and the f l a s k r o t -ated. The deleaded s o l u t i o n was t e s t e d f o r an excess o f ox-a l a t e by adding 1 drop o f d i l u t e d l e a d acetate. Toluene was dropped on the surface of the s o l u t i o n , and the l i q u i d allowed to stand u n t i l c l e a r o f p r e c i p i t a t e d l e a d o x a l a t e . A f t e r de-oantation i n t o a dry f l a s k , the s o l u t i o n was analyzed f o r sucrose and i n v e r t sugars. INVERT SUGARS Dup l i c a t e 5 ml. a l i q u o t s o f the e x t r a c t were used f o r i n v e r t sugar a n a l y s i s . The d i l u t i o n f a c t o r i s 50. The Somogyi-Shafd!'er-Hartmann method des c r i b e d p r e v i o u s l y was em-ployed. (25) I n v e r t sugars are expressed on a percent dry 35, weight b a s i s o f the o r i g i n a l dry powder. SUCROSE Five ml. o f the d i l u t e d e x t r a c t used f o r i n v e r t sugar a n a l y s i s was p i p e t t e d i n t o a 100 ml. volumetric f l a s k and made to volume w i t h d i s t i l l e d water. One^half ml. of concentrated s u l f u r i c a c i d was added and 2 drops of methyl red I n d i c a t o r . The e x t r a c t was hydrolyzed by a u t o c l a v i n g f o r 5 minutes a t 5 pounds pressure; The'hydrolyzed e x t r a c t was n e u t r a l i z e d w i t h 9.8 N NaOH and 51ml• . dupMoate^aliquoTis".usedllniinvert,sugar determination by the somogyi method. The t o t a l d i l u t i o n f a c t o r i s 1025. T o t a l sugars equal sucrose i n v e r t e d p l u s o r i g i n a l i n -v e r t sugars. T o t a l i n v e r t sugar minus o r i g i n a l I n v e r t sugar m u l t i p l i e d by 0.95 equals sucrose present. T o t a l a v a i l a b l e carbohydrates c o n s i s t s o f t o t a l sugars p l u s s t a r c h - d e x t r i n s . The a d d i t i o n o f t o t a l sugars and s t a r c h - d e x t r i n s i s considered as being t o t a l a v i a l a b l e carbohydrates. T o t a l carbohydrate i s expressed as percent of rdegh weight, sucrose and I n v e r t sugar values f o r a l c o h o l e x t r a c t e d pulp are based on a per-cent of dry weight b a s i s of the o r i g i n a l d r i e d powder. IT, ANALYTICAL METHODS - ALCOHOL EXTRACTED PULP STARCH-DEXTRINS The s t a r c h - d e x t r i n f r a c t i o n o f the e x t r a c t e d residue was determined by the method of Loomis and S h u l l . (£7). 36. Malt d i a s t a s e s o l u t i o n was employed. The method i s as f o l l o w s ; D u p l i c a t e 1 gram p o r t i o n s of r e s i d u e from the s o x h l e t e x t r a c t -i o n are t r a n s f e r r e d to 250 ml. f l a s k s ; 50 ml of d i s t i l l e d water use added and the -flasks heated f o r 30 minutes i n a b o i l i n g water bath. The g e l a t i n i z e d s t a r c h s o l u t i o n i s cooled to 35° G. and 10 ml. of a 0.5 percent malt d i a s t a s e s o l u t i o n added along w i t h s e v e r a l drops of toluene. The f l a s k I s h e l d at Get 35° G. o v e r n i g h t , and r o t a t e d o c c a s i o n a l l y i f p o s s i b l e . ,Following i n c u b a t i o n , the e x t r a c t s are f i l t e r e d i n t o 100 ml. f l a s k s , the residue washed, and 2 ml. o f s a t u r a t e d n e u t r a l l e a d acetate s o l u t i o n run i n t o each. The s o l u t i o n s are made to volume and mixed. Deleading i s done by adding 0.3 grams of potassium o x a l a t e to the s o l u t i o n s , which are again f i l t e r e d . 100 ml. o f the c l e a r e d and deleaded e x t r a c t s are hydrolyzed by a u t o c l a v i n g f o r 60 minutes at 15 pounds pressure w i t h 5 ml. o f concentrated HCl. The a c i d i c s o l u t i o n s are n e u t r a l i z e d w i t h NaOH to methyl red I n d i c a t o r , and the t o t a l volume determined. Duplicate 5 ml. a l i q u o t s are used f o r i n v e r t sugar determin-a t i o n by the S&S method. The blank c o n s i s t s o f 50 ml. o f 0.1 percent malt d i a s t a s e s o l u t i o n which i s hydrolyzed as f o r s t a r c h and the amount o f i n v e r t sugar present determined, s t a r c h i s expressed as percent of dry weight o f the erT^taotj une.x-tracked pulp u s i n g the f a c t o r 0.90. INSOLUBLE NITROGEN Dupplioate 1 gram p o r t i o n s o f the e x t r a c t e d residue were a c c u r a t e l y weighed and analyzed f o r i n s o l u b l e n i t r o g e n by the standard K j e l d a h l method, (£3).. The method i s as f o l l o w s : One gram o f dry powder i s a c c u r a t e l y weighed on a 7 cm. f i l t e r paper. The paper i s f o l d e d and placed i n an 800 ml. K j e l d a h l f l a s k . A s e l e n i z e d c r y s t a l i s added, and 25 ml. o f concentrat-ed s u l f u r i c a c i d . E i g h t grams o f a sodium sulf a t e - c o p p e r s u l -f a t e mixture anse s t i r r e d w i t h the a c i d and sample. Heat i s ap p l i e d g e n t l y u n t i l f r o t h i n g i s over and then s t r o n g l y u n t i l c l e a r . Heat i s continued f o r 30 minutes, when the f l a s k i s removed from the burner rack and t r a n s f e r r e d to the fume c l o s e t . A f t e r - c o o l i n g , 250 ml. of water, 100 ml. of 33 percent NaOH, and s e v e r a l pieces of z i n c metal are» placed i n the f l a s k . D i s t i l l a t i o n i s c a r r i e d out s i m i l a r l y as f o r t o t a l n i t r o g e n . One ml. of 0.14 N NaOH i s eq u i v a l e n t to 1 mgm. o f n i t r o g e n . One gram o f sucrose i s used i n place o f the powder f o r the blank. I n s o l u b l e n i t r o g e n i s expressed as percent o f dry weight of the ©]m<$fo-*i 4K&4cvf **>&e-*rjlv.. SOLUBLE NITROGEN The d i f f e r e n c e between t o t a l n i t r o g e n and i n s o l u b l e n i t r o g e n was taken to represent s o l u b l e n i t r o g e n . 38. i&. RESULTS The f o l l o w i n g observations of the e x t e r n a l e f f e c t s of f r e e z i n g and of l e a f regrowth r a p i d i t y were noted; Immediately f o l l o w i n g f r e e z i n g , the lea v e s and p e t -i o l e s became qu i t e b r i t t l e , i n d i c a t i n g complete f r e e z i n g o f the t i s s u e s . F i r s t day - The o l d e r leaves were dead and dark green i n c o l o r . Their p e t i o l e s were thoroughly weakened and drooped. The newer leaves o f 1 i n c h h e i g h t i n the centre of the beet crown had withstood f r e e z i n g . Fourth day - The frozen leaves were withered and no regrowth observed on these beets. Regrowth from the crown centre was beginning on the d e f o l i a t e d beets. r E i g h t h day - No pathogens were present on the de-crowned beets. Rapid regrowth was o c c u r r i n g on a l l other treated beets. Regrowth was removed from the c o n t i n u a l l y de-f o l i a t e d beets. Eleventh day - New leav e s were c o n t i n u a l l y appearing. S i x t e e n t h day - The new leaves had grown s t r o n g l y ( 8 inches i n h e i g h t ) and regeneration o f the f o l i a g e was almost complete. Twentieth day - Normal top appearance and complete regrowth was observed. The c o n t i n u a l l y d e f o l i a t e d beets were s t i l l producing abundant l e a f shoots. The decrowned beets had a t h i c k l a y e r o f wound t i s s u e under the wax on t h e i r c ut sur f a c e s . Thus, i t appears t h a t the g r e a t e s t regrowth o f new leaves occurs from 4-17 days a f t e r f r e e z i n g , and the l a r g e s t sugar l o s s e s would be expected during t h i s p e r i o d * The weather c o n d i t i o n s , o f warm day and n i g h t temperatures f o l l o w -i n g f r e e z i n g i n t h i s experiment were extremely favourable f o r sugar l o s s and l e a f regrowth* N a t u r a l l y o c c u r r i n g f r o s t s might i n c r e a s e i n t h e i r s e v e r i t y a f t e r the i n i t i a l f o l i a r f r e e z i n g on the p r a i r i e s , thus producing a s e t o f c o n d i t i o n s which were not present I n t h i s experiment. New l e a f regrowth would be p a r t i a l l y check-ed by r e c u r r i n g f r o s t s and the sugar l o s s e s obtained would normally not be as l a r g e as those produced by a s i n g l e k i l l i n g f r o s t and an immediate warm-up p e r i o d . RESULTS OF ANALYSES DRY WEIGHT A decrease i n percent dry weight occurred i n a l l treatments. The l a r g e s t decrease was observed on the 16th day a f t e r treatment. The T/C values f e l l I n the f r o z e n beets from 108.0 on the f i r s t day to 80.0 on the 20th day; i n the d e f o l i a t e d beets from 99.0 to 79.0; i n the c o n t i n u a l l y de-f o l i a t e d beets from 96.0 on the 4 t h day to 78.0 on the 20th day; i n the decrowned beets from 91.0 to 77.0 on the 1 s t and 20th days r e s p e c t i v e l y . The;s© decreases as a percentage based on T / C values are: Frozen 26.0 percent; d e f o l i a t e d 20.2 40. percent; continually defoliated 18.7 percent; decrowned 15.5 percent. The highest loss in percent dry weight occurred i n the frozen beets and the least i n the decrowned beets, percent-age losses i n the defoliated beets are similar to those for the frozen beets. The control beets showed no decided reduction in dry weight during the 20 day period. Dry weight results are l i s t e d i n Table III. NITROGEN Total, soluble and insoluble nitrogen percentages are given in Table IV. Total Nitrogen - The values i n a l l treatments de-creased from the 1st to the 8th day. The tendency after the 8th day was towards an increased amount of nitrogen measured as total nitrogen. The T/C values rise above 100 for the frozen, continually defoliated and decrowned beets. Total nitrogen in the control beets varied considerably i n amount. Insoluble nitrogen - The values do not reveal many conclusive changes i n insoluble nitrogen content i n the treated beets. There was a 1st to 8th day decrease In i n -soluble nitrogen following treatment i n the decrowned and de-foliated beets. No general trends i n values were noted after the 8th day. TABLE III PERCENT 'DRY WEIGHT CONTENT OF TREATED AND CONTROL SUGAR BEETS DAYS AFTER TREATMENT > FROZEN % T/C 1 DEFOLIATED % T/C CONT. DEFOLIATED % T/C - CROWN %. T/C CONTROL % 1 21.7 108 20.1 99 mm 18.4 91 20.2 4 20.7 104 19.7 98 19.3 96 19.6 98 20.0 8 17.8 89 18.0 90 18.5 93 17.8 89 19.9 11 18.5 89 18.3 88 17.5 84 16.2 78 20.9 16 16.0 82 14.9 76 17.0 87 15.4 79 19.6 20 17.7 80 17.3 79 17.2 78 16.9 77 22.0 \ DAYS AFTER TREATMENT . FRESH WEIGHT (26 GRAMS) BEET PULP EXPRESSED IN GRAMS DRY WEIGHT FROZEN T/C DEFOLIATED T/C CONT. DEFOLIATED T/C - CROWN T/C CONTROL 4 5.40 102 5.06 97 5.01 96 5.05 97 5.20 8 4.62 91 4.68 92 4.80 95 4.63 91 5.08 11 4.80 89 4.76 88 4.55 84 4.21 78 5.41 16 4.16 82 3.87 77 4.42 88 4.00 79 5.05 20 4.60 80 4.50 79 4.47 78 4.39 77 5.71 1 - T/C : PERCENT IN TREATED/PERCENT IN CONTROL X 100 8 I t A i \\ 1 • - — N , \ / \ l \ \ V t \ A' 7 / / \ \ K i v V - / / / \ 1 l _ / it w 1 I _\ A' // / \ V \ // \ \ \ / \ \ \ / 1 | i i 6 E: 2 o /( b 1 Zo R.OZ N DEF "CD a i A ft i T / i i f-V± // / _ / / \ / \ -/ / \ \ /— / \ / _ l 1- • '\ 1 U i i - •I- 1 o. 1 , 7 - | - ' — | \ — - \ / / \ v- —y~ \ f-\ \ \ -— u. i 1 3 .-< # D l I O 1 1 D.E.CRO It D c fONT r r -- - - i - a EI I I" 1 / - -\ is t 1 1 1 1 | 41. Soluble nitrogen - There was a decrease In soluble n i t r o -gen i n a l l cases from the 1st to the 8th day. After the 8th day, the percentage of soluble nitrogen i n the treated beets was generally higher than i n the control beets. The defoliated beets were an exception to this. The T/C values f e l l and rose as follows: values for the 1st, 8th, and 20th days - frozen 102.0, 45.0, 131.0; defoliated 87.0, 40.0, 53.0; continually defoliated 93.0 (4th day), 74.0, 106.0; decrowned 110.0, 85.0, 138.0. SUCROSE The average percentages of sucrose based on fresh weight were: control 13.4; frozen 13.1; defoliated 12.3; con-tinually defoliated 12.5; decrowned 12.9. The values obtained when the percent sucrose fresh weight i s converted to percent on a dry weight basis show that, expressed as T/C, the values increased i n the frozen beets from 101.0 on the 4th day to 120.0 on the 20th day. The other groups had the following T/C results for the same days: defoliated 96.0 to 123.0; con-tinually defoliated 94.0 to 119.0; decrowned 111.0 to 121.0. Calculation of percent sucrose dry weight was done by deter-mining the percent dry weight of the 26 gram pulp portions used for sucrose analysis and converting percent sucrose fresh weight to percent sucrose dry weight for each individual analysis. The sucrose values for the alcohol extracts indicate that a sugar loss has occurred i n the treated beets. The TABLE IV TOTAL, SOLUBLE AND INSOLUBLE NITROGEN CONTENT OF TREATED AND CONTROL SUGAR BEETS DAYS AFTER TREATMENT  TREATMENT CONTROL FROZEN DEFOLIATED X XX XXX TOT INSOL SOL TOT T/C INSOL T/C SOL T/C TOT T/C INSOL T/C SOL T/C - % - fo. • - . % - % • -• - • - -1 4.94 .423 4.52 5.00 101 .397 94 4.60 102 4.37 89 ,441 104 3.93 87 4 4.63 .362 4,27 3.63 79 .416 115 3.21 75 4.00 86 .333 92 3.67 86 8 5.00 .392 4.61 2.37 47 .303 77 2.07 45 2.12 42 .287 73 1.83 40 11 3.31 .345 2.96 5.00 151 .369 107 4.63 156 5.48 165 .338 98 5.14 174 16 4.06 .435 3.62 6.10 150 .407 94 5.69 157 3.88 96 .414 95 3.47 96 20 3.56 .355 3.20 4.56 128 .375 106 4.18 131 2.00 56 .311 88 1.69 53 x - TOTAL xx - INSOLUBLE IN 70% ETHANOL xxx - SOLUBLE (TOTAL-INSOLUBLE) TABLE IV (CONT'D) TOTAL, SOLUBLE AND INSOLUBLE NITROGEN CONTENT OF TREATED AND CONTROL SUGAR BEETS DAYS AFTER TREATMENT  TREATMENT CONTINUAL DEFOLIATION DECROWNED TOT. T / C 1 INSOL. T/C SOL. *-T/C TOT. • T/C INSOL. T/C SOL. T/G 1 - - - - - - 5.37 108 .394 93 4;98 110 4 4.31 93 .411 114 3.90 91 3.94 85 .357 99 3.58 84 8 3.69 74 .435 111 3.25 70 4.25 85 .315 81 3.93 85 11 3.50 106 .406 118 3.09 104 4.94 149 .431 125 4.51 152 16 3.88 96 .343 79 3.54 98 4.69 116 .363 84 4.33 120 20 4.81 135 .322 91 4.49 140 4.75 133 .314 88 4.44 138 1 " T/C : PERCENT TREATED/PERCENT CONTROL 42. results based on dry weight indicate that the decrowned beets lost the highest percent of sucrose. The continually de-foliated beets showed the next highest loss followed by the defoliated and frozen beets* The percentages of sucrose based on dry weight in the alcohol extracts are lower than those obtained when peroent sucrose fresh weight i s expressed on a dry weight basis* sucrose results are given i n Table V* INVERT SUGARS Invert sugars, as analyzed for i n fresh pulp and expressed in percent dry weight, increased i n amount follow-ing a l l treatments* A noticeable rise i n invert sugars occurred after the 8th day* The T/C values on the 1st, 8th, and 20th days were: frozen 87*0, 116*0, 140*0; defoliated 102, 138*0, 1*6*0; continually defoliated 104*0, 136*0, 262*0; TABLE V SUCROSE CONTENT OF TREATED AND CONTROL SUGAR BEETS - AQUEOUS EXTRACT TREATMENT DAYS AFTER ~ TREATMENT CONTROL FROZEN DEFOLIATED CONTINUALLY DEFOLIATED DECROWNED fo FRESH WT. % T/C fo T/C f0 T/c fo T/C Q_ e m m • m « # w • 4 13.20 13.90 105.0 12.30 93.2 12.00 91.0 14.20 108.0 8 13.90 13.20 95.0 12.00 86.5 12.00 86.5 13.20 95.0 11 13.90 13.20 95.0 13.90 100.0 12.50 90.0 13.20 95.0 16 13.20 12.50 94.8 10.80 81.8 13.90 105.0 12.00 91.0 20 12.90 12.50 97.0 12.50 97.0 12.00 93.0 12.00 93.0 fo DRY WT.l fo T/O fo TJG fo T/C % ~ T/C 4 66.1 67.0 101 63.4 96 62.3 94 73.2 111 8 71.2 74.3 104 66.7 94 65.0 91 74.2 104 11 66.8 71.5 107 76.0 114 71.4 107 81.5 122 16 68.0 78.2 115 72.5 107 82.0 120 78.0 114 20 58.8 70.7 120 72.2 123 69.8 119 71.1 121 1- PERCENT SUCROSE FRESH WEIGHT CONVERTED TO PERCENT SUCROSE BASED ON THE DRY WEIGHT OF 26 GRAMS OF PULP. TABLE V (CONT'D) SUCROSE CONTENT OF TREATED AND CONTROL SUGAR BEETS - ALCOHOL EXTRACT DAYS AFTER TREATMENT CONTROL FROZEN DEFOLIATED CONTINUALLY DEFOLIATED DECROWNED % PF.DRI^ WT;- % T/C % T/C % T/C % T/C 1 79.0 75.5 96 67.0 85 - 67.8 84 u 73.3 77.9 106 76.4 104 60.7 83 68.5 94 8 66.0 65.2 99 76.3 116 61.7 93 64.8 98 11 68.9 61.8 89 64.4 94 59.0 .86 57.7 83 16 67.4 61.7 92 45.8 68 60.4 90 59.3 88 20 71.6 58.8 82 60.8 85 62.8 88 55.0 77 1 - PERCENT DRY WEIGHT OF ORIGINAL DRIED PULP. e & i _ - 1 i — \ i / / V V // /-A \ J \\ // 1 / \\ 1 / > / J 1 \ 1 \ f I -''I — / / 1/ / / —t \ / /-/ \ - / c \ / — ---- - -* - I lie 2 o z a F R o z. E ) A T t - r> s> g. y L1 i I — — - i I 1 / '/ / -i k \ \ A ' i \. /I ' — .1 - i 1 f 1 1 I i i a< i IT 1 / / V 1 / \ f / / \ \ --• — ' - - -1 1 1 1 1 i Zo 2( 0 •\ | .t= _v S.E a Ca 1 P 0. A T f V u l- T f > f A r.l ,0 1 "| ' < t TV. •= -1 JUL&J | c _ALLC R M > I I 1 I ! I I 1 i i i i decrowned 117.0, 116.0, 232.0. The p e r c e n t o f i n v e r t sugars i n the c o n t r o l b e e t s remained f a i r l y c o nstant. The g r e a t e s t ! r i s e i n i n v e r t sugars was found i n the decrowned b e e t s , w i t h the d e f o l i a t e d beets showing the l e a s t r i s e i n i n v e r t s . I n v e r t sugars, as determined by a n a l y s i s o f the a l c o h o l e x t r a c t o f the d r i e d beet p u l p , were i n c r e a s e d over the c o n t r o l s f o r the l a t t e r 16 days i n the decrowned and con-t i n u a l l y d e f o l i a t e d b e e t s . The i n v e r t sugar content o f the fr o z e n and d e f o l i a t e d beets i n c r e a s e d to the 4 t h day and then a s l i g h t decrease i s noted. In a l l analyses, whether made on the f r e s h pulp or dried, p u lp, the average percentage o f i n -v e r t sugars i n the t r e a t e d beets i s g r e a t e r than t h a t o f the c o n t r o l b e e t s , i n v e r t sugar v a l u e s are g i v e n i n Table VI. STARCH-DEXTRINS The values obtained f o r the s t a r c h - d e x t r i n f r a c t i o n o f the e x t r a c t e d beet pulp do n o t i n d i c a t e any q u a n t i t a t i v e changes which have o c c u r r e d i n r e l a t i o n to the treatments. A g e n e r a l i n c r e a s e or decrease i n the s t a r c h - d e x t r i n f r a c t i o n i s n ot noted i n these r e s u l t s . The percentage o f s t a r c h i n sugar beet r o o t i s no r m a l l y low. s t a r c h - d e x t r i n v a l u e s are give n i n Table V I I . TOTAL CARBOHYDRATES A decided decrease i n t o t a l carbohydrates o c c u r r e d i n a l l treatments. The lowest drop was found i n the d e f o l -TABLE VI > INVERT SUGAR CONTENT OF TREATED AND CONTROL SUGAR BEETS - AQUEOUS EXTRACT DAYS AFTER TREATMENT TREATMENT , CONTROL FROZEN DEFOLIATED CONT. DEFOLIATED DECROWNED % FRESH WEIGHT 1 % T/C % T/C % T/C % T/C 1 - m - *• - - m -A 0.057 0.051 89 0.057 100 0.057 100 0.065 I H 8 0.071 0.054 76 0.061 86 0.069 97 0.054 76 11 0.055 0.057 103 0.067 122 0.063 114 0.050 91 1 6 0.063 0.069 111 0.053 8A 0.061 97 0.110 174 20 0.058 0.065 112 0.053 91 0.120 207 0.100 173 % OF DRY WT.2 % T/C % T/C % T/C % T/D 4 0.284 0.248 87 0.288 102 0.294 104 0.332 117 8 0.360 0.304 85 0.338 94 0.372 104 0.304 84 11 0.266 0.307 116 0.368 138 0.362 136 0.308 116 1 6 0.325 0.430 132 0.354 109 0.358 109 0.748 230 20 0.264 0.368 140 0.306 U6 0.690 262 0.610 232 1 - PERCENT OF FRESH WEIGHT OF ORIGINAL FRESH PULP. 2 - PERCENT DRY WEIGHT OF 26 GRAMS OF PULP. TABLE VI (CONT'D) INVERT SUGAR CONTENT OF TREATED AND CONTROL SUGAR BEETS - ALCOHOL EXTRACT DAYS AFTER TREATMENT TREATMENT CONTROL % DRY WEIGHT 1 FROZEN % T/C DEFOLIATED % T/C CONT. % DEFOLIATED T/C DECROWNED % T/C 1 2.1 1.8 2.0 95 - - 1.9 91 A 1.8 3.1 172 2.2 122 3.1 172 2.3 128 2.9 4.1 HI 1.1 38 3.1 107 4.2 H5 11 2.7 3.4 126 4.1 152 3.6 133 4.2 158 16 2.8 3.3 118 3.7 132 2.8 100 4.1 H6 20 3.0 3.6 120 3.8 127 3.4 113 4.4 H7 1 - PERCENT DRY WEIGHT OF ORIGINAL DRIED PULP -— / t I s A, \ 1 i i / / i / / \ —/ \ \ -' '• t /• / _ — 1 I ~~\ I I V, 1 1 1 • 1 t > \ \ // \, \ A. // 7 r \ 7 \ I \ \ 1 1 \ It, 7( \ \ b I ,0 / J F * .0 L r i - - »-• — ~ / . / 8 4 \ i I / I i \ •i A t \ \ f y J \ / / \ \ / / f X / / / j / 1, i \ • < 1 1 T/ i 1 o o -< •o >_( Zt _ > \ V -1A \ V ~y \ / \ -<-\ \ J • A — l<o Zc > I H 9 -' 0 \ f \ - H - e ^ r-t -G — 1 1 | 1 D i e ..f ML 1 Eh Co r_ _ Lf X n •J X 1 A =--1 o 0--r f J -< Jl T.. ) c l_l __ A L C O H O L E X T R A C T ] ) TABLE VII STARCH - DEXTRIN CONTENT OF TREATED AND CONTROL BEETS DAYS AFTER TREATMENT CONTROL % DRY WEIGHT1 FROZEN % T/C DEFOLIATED % T/C CONT. DEFOLIATED DECROWNED % T/C % T/C 1 0,528 0.528 100 0.383 73 - - 0 . 4 4 0 84 4 0.309 0.445 144 0.258 84 0.437 1 4 2 0.430 139 8 0.620 0.388 63 0.308 50 0.237 38 0.210 34 11 0.297 0.424 142 0.470 158 0.490 165 0.325 109 1 6 0.485 0 . 4 2 0 87 0.425 88 0.354 73 0.405 84 20 0.609 0.479 79 0.490 81 0.400 66 0.425 70 1 - PERCENT DRY WEIGHT OF ORIGINAL DRIED PULP. •44. iated beets, while the decrowned beets have the highest drop. Total carbohydrate values are given i n Table VIII. fne values qre based on alcohol extract ion-ENZYMES PHOSPHORYLASE In the second experiment of October. 1954, phospho-rylase activity appeared to decrease with time i n a l l treat-ments. Activity i n the control beets, especially from the 11th day was considerably higher than in the treated beets. There was a sudden decrease i n phosphorylase activity from the 1st to the 4th day i n the treated beets. The results of experiment II are given i n Table x» PHOSPHATASE Phosphatase activity decreased from the 1st to the 11th days i n a l l treatments except decrowned. An increase i n -activity occurred between the 11th and 20th days. In the decrowned beets, phosphatase activity Increased slightly to-wards the 4th day, then decreased u n t i l the 16th day. The micrograms of phenol values on the 1st, 11th, and 20th days respectively for each group were: frozen 550, 340, 460; de-foliated 520, 350, 440; continually defoliated 480(4th day), 350, 440; decrowned 530, 380, 410; control 450, 370, 450. Although the activity of phosphatase f e l l i n the control beets, the decrease i s not as-high as that in the treated beets. The results are given in Table IX. TABLE VIII TOTAL CARBOHYDRATE ( SUCROSE, STARCH-DEXTRINS, INVERT SUGARS) CONTENT OF TREATED AND CONTROL SUGAR BEETS DAYS AFTER TREATMENT LTMENT CONTHOL % DRY WEIGHT 1 FROZEN % T/C DEFOLIATED % T/C CONT. % DEFOLIATED T/C DECROWNED % T/C 1 ' 81.6 77.8 95 69.4 85 mm - 69.1 85 4 75.4 81.4 108 78.9 104 64.3 86 71.2 95 8 69.5 69.7 100 77.7 112 65.0 94 69.2 96 11 71.9 65.6 92 69.0 96 63.1 88 62.2 87 16 70.7 65.4 93 49.9 71 63.6 90 63.8 90 20 75.2 62.9 84 65.1 87 66.6 89 59.8 79 1 - PERCENT DRY WEIGHT OF ORIGINAL DRIED PULP - BASED A U O O H O L . EXTRACTION 1 b u -M \ h -/ \\ vr \ \ / // V / " \ \ \ f \ \ -_ 4- / • l _ /'I 1 \ Is //-I 1 1 L V 1 f \ \U /- u / —^ " / \ / / / / \^ --\ I 1 1 1 i lb 2o ib zo La. a. Z . . E . N D I. L( . . i r_ ) © 8 I 1 .1 1 / • I • - --// \\ — /'/ V ./. / V / f \ / / / h -/ 1 \ _ l i / J V 1 1 \ i i I '/ I 1 \ \ / \ - 1 / L\, '- • V ~ / — / - \\ \ \L\ / N L. — N r i i i I I I ! .1 i 1 i 1 Z O lla to 1 — — - 7, t ' T •a e M E D. - D.E O- N E. • c o N r bF Q —1— A T _E D T • FZHT —\— 1 Cit A C T L AK O O HY a TABLE IX ROOT ENZYME ACTIVITIES OF TREATED AND CONTROL SUGAR BEETS - EXPERIMENT I TREATMENT ENZYME DAYS AFTER TREATMENT FROZEN_T/C DEFOLIATED T/C CONT.DEFOL T/G - CROWN T/C CONTROL ACT. 1 AAGT. ACT. ACT. AGT. 1 550 125 520 116 - " 530 118 450 4 500 112 480 107 480 107 540 120 450 8 430 98 450 102 430 98 400 91 440 PHOSPHATASE 1 ± 3 4 0 9 2 350 95 350 95 380 103 370 16 400 100 410 103 430 108 360 90 400 20 460 102 440 98 440 98 410 91 450 5To3 xTo3 5To3 xTo~3 xlo~3 1 0.49 197 0.40 160 - - 0.60 240 0.25 4 0.16 41 0.18 46 0.23 59 0.34 87 0.39 8 0.32 140 0.27 118 0.37 160 0.21 91 0.23 B " AMYLASE H 0.28 61 0.28 61 0.23 50 0.33 72 0.46 16 0.30 40 0.33 44 0.35 44 0.49 65 0.75 20 0.18 60 0.28 93 0.42 140 0.33 110 0.30 1 - ENZYME ACTIVITY TABLE IX (CONT'D) ROOT ENZYME ACTIVITIES OF TREATED AND CONTROL SUGAR BEETS - EXPERIMENT I TREATMENT ENZYME DAYS AFTER TREATMENT FROZEN T/C DEFOLIATED T/C CONT.DEFOL T/C - CROWN T/C CONTROL ACT. 1 ACT. ACT. ACT. ACT. x l O 2 x l O 2 x l O 2 x l O 2 x l O 2 1 1.37 120 1.30 114 - - 1.41 123 1.15 4c 1.23 126 1.24 128 1.39 143 1.09 113 0.97 8 1.05 130 1.08 133 1.10 136 0.72 89 0.81 CATALASE 11 0.9 7 79 0.74 60 1.08 88 1.0 7 8 7 1.23 16 0.81 58 1.11 71 1.31 85 1.20 78 1.55 20 1.05 84 0.66 53 0.98 79 1.12 89 1.25 1 - ENZYME ACTIVITY D 1, 1 o -1— t — \ // —> // K h \ ' /'/ H i \ u '/ \ / /•/ —\ -\-V h / •v v. l_ 1 ^ _4-_ i // V" 1 \ \ r 1 r \ \ 1 A -t / /I / \\ / / \\ / J /> \\ y A ' f / \\ 1 - 'P \<o z O Z O Ff >.z ._e.r> D t F o. _E .0 1 O T \ ft t 1 — t 1 1 i _ 4 /• ' / \ / // \ /> /y \ \ / r V // _ \ f - / l \ t A 1 \j 1 © j _ /[ ,' v" / \ / \ // 'I " x~ r / \ i 1 1 — lb Ik j 20 1 D.E..CR.C /. Sl.t.O n X 5.1 .1 1 1 - c r \ - - /c - i -r- E- >-D X e t J : r: • 7( - C O A C l_ \/_ i" t / B \ b i 1 i \ i / N — i J }/ \ \ \ \ A-i. \ —^ 1 A I _ i i 1 \ 1 \ A V • 1 1 1" • — \ N / i / y— \ -V - \ — - V • 1 1 i lb to lb -_ o z £ 0.e _C r D i 5 p — ) i — i ft A I 1 \ i i — / L L J i f V 7" i \ V \ r \ /1 ~t i / 1-\ \ • i 1 1 1 II 1 c Z-< -\ / - A -V A / \ jIS // \ Sr A \ •- \ --- -• - — 1 1 | 1 lb 2 0 \z _! <-X o D 1 | | 1 Die . . R . C >a£..d> Cc * -T D e £ L . I v i •- .1. 5< Q 1 1! :t - T T 1 E -AT V 4-5 BBTA-AMYLASB No changes occurred for beta-amylase activity which could be definitely attributed to starch degradation. The values found i n both experiments (August and October) are not similar. There was a tendency for beta-amylase activity i n the treated beets to be lower than that of the controls i n ex-periment I but this relationship was not duplicated in the 2nd experiment* The results of experiment I are given i n Table IX, and those of experiment II i n Table X. CATALASB Catalase activity increased i n the frozen beets from the 1st day to the 11th day and then decreased towards the 16th day. The pattern in the defoliated beets was similar to that for the frozen beets - that Is, an Increase i n activity to the 11th day following treatment and then a general de-crease. In the cohtinously defoliated beets, the catalase activity on the 4th and 8th days was higher than i n the cont-rol beets. There was also a decrease in activity from the 11th to the 20th day in this group* The activity decreases as would be expected, i n the decrowned beets. The results are given i n Table IX* iNYERTASB $he results of experiment II are given i n Table X* Invertase activity increased i n the frozen beets and decrowned beets from the 1st to the 8th day after treatment. Activity 46 of the enzyme on the 15th day was greater In a l l treated beets than i n the controls* Invertase activity could not be detected i n the continually defoliated beets on the 1st and 12th days following treatment. Activity i n both groups of defoliated beets was generally lower than i n the control beets* TABLE X ROOT ENZYME ACTIVITIES OF TREATED AND CONTROL SUGAR BEETS - EXPERIMENT II ENZYME DAYS AFTER TREATMENT FROZEN ACT. T/C DEFOLIATED ACT. TREATMENT T/G CONT. DEEOL ACT. T/G - CROWN ACT. T/C CONTROL ACT. 1 130 100 90 70 0 0 170 131 130 A 610 185 830 252 390 118 370 112 330 INVERTASE 8 810 262 290 93 190 61 420 136 310 12 170 85 140 70 0 0 110 55 200 15 240 218 360 326 240 218 560 520 110 1 56 400 72 515 69 490 33 236 14 4 34 74 17.5 38 21 A6 13 28 46 PHOSPHORYLASE 8 65 52 17.5 14 164 131 72 57 126 12 25 23 65 59 71 65 53 48 110 15 133 88 24 15.9 86 57 69 46 152 1 - ENZYME ACTIVITY TABLE X (CONT'D) ROOT ENZYME ACTIVITIES OF TREATED AND CONTROL SUGAR BEETS - EXPERIMENT II ENZYME BETA AMYLASE DAYS AFTER TREATMENT LTMENT FROZEN ACT. 1 xlO^ T/fc DEFOLIATED ACT. xL03 T/C CONT. DEFOL. ACT. xlO^ T/C - CROWN ACT.. xl03 T/C CONTROL ACT. xlO? 1 0.28 65 0.29 67 0.39 91 0.34 79 0 .43 4 0.32 266 0.30 250 0.27 225 0 .32 266 0.012 8 0.23 9 2 0.16 6A 0.012 - 0.12 4 8 0.25 12 o.u 5 2 0.23 85 0.25 92 0.27 100 0.27 15 0.28 108 0.29 112 0.33 127 0 . 3 4 131 0.26 1 - ENZYME ACTIVITY 47 INVERTASE ACTIVITY IN VARIOUS REGIONS OP THE BEET ROOT The highest activity value found was In the crown portion of the beet root. Invertase acti v i t i e s i n other regions in order of decreasing activity were: beet centre 290; outer surface 279; beet tip 210. The crown had an act* i v i t y of 300 E.A. units. The values refer to milligrams of Invert sugar difference between the actual run and the blank multiplied by 1000. The results are l i s t e d i n Table I. VALIDITY OF THE SAMPLING METHOD These results showed that the 40-gram aliquot of beet pulp which was drawn from the total weight of pulp con-stituted a representative sample. The values obtained for each aliquot agree closely with one another. The tttH value for these results i s small, indicating that the sampling error i s negligible. The results are given i n Table I I . TOP WEIGHTS AND ROOT VDEIGHTS AS RELATED TO SUGAR CONTENT No correlation could be found when the average top weights of each group of defoliated beets were compared to the average root weights and sugar content of those groups. Also, no correlation could be found between percent sugar and average root weight, some typical results are given i n Table XI* TABLE XI COMPARISON OF AVERAGE LEAF WEIGHTS TO AVERAGE ROOT WEIGHTS AND SUGAR CONTENT AT .HARVEST TIME (RESULTS GIVEN FOR DEFOLIATED GROUP) pay after Average1 Average Average wt. % sucrose treatment leaf wt. root wt. of regrowth fresh wt* 1 415 gms 180 gms n i l . -4 422 n 474 II n i l 12*30 8 471 N 168 n 20 gms 12.00 11 431 n 144 » 134 » 13*90 16 509 n 160 it 145 '« 10*80 20 405 n 173 it 148 12*50 1 - AVERAGE OF 10 BEETS* 48. 9, DISCUSSION Tne results of this experiment indicate that there i s no relation between average leaf weight and average sucrose content. Also, there i s no relation between average root weight and average sugar content in mature beets* Both leaf and crown removal cause a pronounced decline i n the yield of dry matter of the root* Tne peroent dry weight decreases with time following treatment* percent dry weight decreases i n the continually defoliated and de-foliated beets are similar, and indicate that the newly formed attached leaves do not constitute the only source of dry weight loss* The defoliated beets exhibited a larger dry weight decrease than the continually defoliated beets* This upholds the view that measurable quantities of photosynthate do not pass into the root before 20 day's time* Frozen beets have the highest dry weight decrease and the second highest sucrose loss of the other treated groups* The following conclusions are made with respect to dry weight decreases i n beet root tissue after f o l i a r loss: 1 * Frost may i n i t i a t e a higher rate of meristernatic activity i n beets, thus increasing leaf regrowth, sucrose loss and dry weight decrease* 2*, A decrease |n dry weight i s either related to a loss of total solids, as sucrose, which i s used i n leaf re-growth, or an increase i n the hydration of the beet root 49. tissue. Dry weight loss i s l i k e l y a result of both a sucrose disappearance and water increase i n the beet root, these sub-stances being u t i l i z e d i n leaf regrowth. 3. Although the decrowned beets have the highest sucrose losses and the lowest dry weight decreases, they have the highest percent of invert sugars. However, the amount of total carbohydrates i s lower i n the decrowned beets. It may be that the low decrease i n dry weight i s an effect caused by the evaporation of moisture from the cut root surface. The average percent fresh weight of sucrose i s lower in each treated group than i n the control, A 0.1 percent sucrose difference can be detected with the polarimeter used in the experiment. The.duplicate analyses consistently checked within 0.1 percent. It i s observed that the T/C values i n a l l treatments rise above 100 after the 8th day when percent sucrose fresh weight i s converted to dry weight percent, percent sucrose, expressed by this method, increases as the percent dry weight decreases. The decreases i n percent sucrose for each period after treatment do not equal the corresponding dry weight loss. This suggests other factors such as .^educed transpiration and increased hydration whioh lower the dry weight percent tof,. the tissue. The averages of the T/C values for the alcohol extract are as follows; frozen 94.0; defoliated 92.0; continually de-foliated 88.0; decrowned 87.0. These results indicate that a 50. sucrose loss has occurred i n the treated beets. Foliar freezing, continual or a single defoliation, and decrowning a l l cause a reduction i n percent sucrose i n the beet root. In this experiment the lowest percent sugar was found to occur between 11 and 20 days after freezing and defoliation, since the most rapid leaf regrowth was apparent during this period i n these beets, a positive correlation between' percent sugar loss and leaf regrowth i s suggested. These results correspond with those of other workers (5) who have noted the relation between leaf regrowth and percent sugar loss. Leaf regeneration was complete at the end of 20 days following f o l i a r loss and i t would be expected that the per-cent of sucrose i n the beet root should rise after this time, a l l other conditions being favourable. There i s no evidence from the results to indicate that sucrose was being stored i n the frozen and defoliated beets on the 20th day. It was noted that new leaf shoots were actively appearing on the continually defoliated beets at the 20 day point. This leads to the conclusion that the region of high-est sucrose disappearance i s in the beet crown, l i k e l y through increased respiration, and that whether or not the new leaves are removed, the meristematie activity i n the crown continues at a higher level than In the normal growing beet. Removing the meristematlc centre or crown from a beet 5 1 • would, I t was thought, reduce sucrose loss considerably, i t was found that sucrose loss in the decrowned beets i s higher than in any other treatment. The invert sugar content i n the decrowned beets was higher on the 8th, 11th, 16th end 20th days than i n the beets treated otherwise. Invertase activity in the decrowned beets was higher than i n the control beets, particularly for the f i r s t 8 days after treatment. An increase i n the amount of invert sugars present occurred following freezing, defoliation or decrowning. Al-though an inversion of sucrose would be accompanied by a rise i n invert sugars, i t may be that the inverts formed are inter-mediate and transitory i n metabolism. The percent increase i n invert sugars does not equal percent sucrose loss, suggesting that the glucose and fructose sugars are u t i l i z e d almost immediately i n leaf regrowth or i n increased respiration with-in certain parts of the beet root - most probably the crown. It has been found that the crown of a sugar beet contains reducing sugar i n concentrations as high as 2 peroent of the dry weighty 2 % T h e concentration of invert sugars found i n the centre of the root i n the alcohol extract analyses were higher than 2 percent of the dry weight. This suggests a slight inversion of sucrose during alcohol extraction. Since the total amount of reducing sugars i s f a i r l y constant throughout the growth of the beet (11), any increase in invert sugars should be attributable to some definite bio-52. chemical change or changes. It i s possible, and supported by other workers (25), that sucrose i s inverted to the reducing sugars which move from the root cells into the phloem and are translocated to the beet crown where they are required i n anabollam involving new tissue formation, i t has been stated that sugar losses have been found where no visible leaf re-growth has occurred (1), This i s substantiated by the sugar loss in the decrowned beets. In a l l other treatments, abundant leaf shoots were seen appearing at 3 days time following treatment. The conclusion i s that sucrose loss i s not necessarily accompanied by visible leaf regrowth. The con-clusion reached from the results of this experiment i s that an increase i n invert sugars does occur following f o l i a r loss, and that i t i s at the expense of the stored sucrose. The percent of starch i n sugar beet root i s low, as the beet synthesizes sucrose as i t s main storage product. The results for "starch-dextrins do not indicate polysaccharide synthesis at the expense of sucrose but the T/C values suggest a loss of starch. The beta-amylase and starch phosphorylase activity in beet root tissue i s low, which might indicate that the starch synthesizing and degradation systems i n sugar beet root are not as efficient as they are in other plant tissues, as for example the potato tuber, Leucoplasts have been found i n beet root c e l l s , and a suggestion has been made (13) that starch synthesis occurs only when sucrose concentrations are relatively high i n the root c e l l s . The conclusion in this 53. experiment regarding starch-dextrins i s that they are of no direct importance to sucrose loss after f o l i a r removal either by frost or manual defoliation. The average percent of dry weight starch-dextrin values i n the controls i s higher than the corresponding average for the treated beets, hence a decrease in the amount of staroh i n the treated beets i s suggested* The decrease in percent of total carbohydrates follows the fact that sucrose disappears, since sucrose loss and invert sugar increase occurs, then not considering starch-dextrin synthesis or breakdown, the percent of total carbo-hydrates would at f i r s t be expected to remain constant* A measurement of total carbohydrates i n beet root tissue when leaf regeneration i s occurring would l i k e l y give a value lower than that for a normal beet since part of the invert sugar present as a result of sucrose inversion i s being used in catabolism. This would lower the amount of carbohydrate in the beet root tissues, hence total carbohydrate estimations provide a reasonable basis for determining both the amount of sucrose loss and the time after treatment when the highest loss occurs. It i s not known from the results of this work in which region of the beet root the highest total carbo-hydrate reduction i s found* An increase i n soluble carbohydrates would favour an increase i n nitrogen i n the beet root* The principle 54. regions of protein synthesis In plants are i n meristems or i n storage tissue, particularly the leaf. Amino acid synthesis may occur i n the roots of some plants. These amino acids may be translocated from the tissues i n which they originate to distant tissues before being converted into proteins, i n meristematic centres, protoplasmic proteins are constructed from amino acids. Amino acid synthesis can not occur without an adequate supply of carbon compounds, primary synthesis of amino acids and similar compounds occurs only at the ex-pense of carbohydrates or their derivatives, which serve to-gether with nitrogen containing compounds as a source of energy. Rapid amino acid synthesis results i n a diminuition i n the amount of carbohydrates i n a plant ( 3 0 ) . Growth of meristems requires carbohydrates and nitro-gen. Both of these kinds of compounds are assimilated i n relatively large quantities, especially during the c e l l division and enlargement phases of growth. Considerable amounts of carbohydrates are also used i n the process of respiration in any actively growing meristerns. From the above discussion, the following nitrogenous and carbohydrate changes might be expected to occur In the beet root following f o l i a r loss and active leaf regrowth; 1. since leaf regrowth requires protein synthesis which in turn requires: amino acid synthesis, then carbohydrate i n the growing plant w i l l be rapidly u t i l i z e d . 55, 2. An Increase In soluble carbohydrates would be accompanied by an Increase In organic nitrogenous compounds* A decrease i n soluble carbohydrates would inhibit organic nitrogen synthesis* 3. An increase i n organic nitrogen could be ex-pected in the crown of the beet which is respiring more than the other portions of the root* An increase In invert sugars occurs i n beets which are in the stage of active leaf re-growth* An invert sugar increase favours amino acid synthesis* 4. A decrease in organic nitrogen could be expected in those regions where carbohydrates are disappearing rapidly* One region i s the centre and outer edges of the beet root. When proteins are degradated, an Increase i n soluble nitrogen would appear and the insoluble nitrogen values would drop. In this experiment the diagonal section across the centre of the beet and not the crown tissue was analyzed for nitrogen. The nitrogen values are not, then, those of the main active growing or leaf producing region of the beet. Total nitrogen decreased from the 1st to the 8th day; in a l l treated beets* The decrease in nitrogen may be associated with a translocation of amino acids out of the region of the beet root analyzed* A measureable rise i n total nitrogen occurred after the 8th day and i t was after this period that the highest percentage of Invert sugars could be detected, with this increase i n soluble, available 56. carbohydrate i n the form of glucose and fructose, amino acid synthesis, as well a3 the synthesis of other nitrogenous compounds i s favoured* The decrease In soluble nitrogen appears to uphold the thought that soluble nitrogenous compounds are trans-located to the active growing regions. Inorganic nitrogen i n the form of nitrate or n i t r i t e s may be translocated to the growing regions. After the 8th day, the percent of soluble nitrogen increased i n a l l beets but the defoliated. This i n -crease In soluble nitrogen may possibly be associated with the observed increase i n invert sugars, such soluble nitro-genous substances commonly found in the beet root as glutamine, asparagine, betain and allantoin may be synthesized. Insoluble nitrogen decreased In amount from the 1st to the 8th day In the _ • defoliated and decrowned beets. Insoluble nitrogen consists of organic nitrogen as proteins. A decrease in insoluble nitrogen would mean an increase i n soluble protein and other nitrogenous compounds as amino acids for example. Thus, a protein breakdown Into the con-stituent amino acids i s suggested, the aminos being trans-located to the beet crown where growth i s occurring. In summary of the nitrogen values noted i n this ex-periment, the root tissue at the centre of the beet loses nitrogenous substances continually u n t i l the 8th day, after 57. whioh time there i s a general increase i n iSV.l;o:bjexa£i1tr^ ..,s-ENZYMES A phosphorylase activity decrease suggests that the - synthesis of starch i s reduced, since starch phosphorylase in sugar beet root l i k e l y has a minor role in total carbo-hydrate metabolism of the tissue, a decrease i n activity would possibly be insignificant, increased respiration due to leaf regrowth would favour the use of glucose-phosphate as a respiratory substrate i n new tissue formation and not starch synthesis* phosphatase activity decreased i n a l l treated beets from the 1st day to the 11th day and Increased between the 11th and 20th days. The action of plant phosphatase i s the splitting off of phosphate from compounds containing phosphate, the end products being inorganic phosphate and a non-phosphate. A common phosphatase reaction i n plants 1st phosphatase gluoose-6-phosphate — — — — > - glucose • Inorganic 4 H2O phosphate This i s an irreversible enzymatic reaction. A decrease i n phosphatase activity would result i n less substrate being s p l i t into inorganic phosphate and glucose. The effect of this i s that more phosphorylated glucose Is available for respiration. The reason for a lower apparent phosphatase activity i n treated beets in t h i s 58. experiment l a not known. It may be associated with an i n -creased respiratory rate. Although beta-amylase activity i n sugar beet root was detected, the activity was very low, requiring incubation of the enzyme with the substrate for a longer period of time than i s normally used in the estimation. No correl-ation can be found between starch-dextrin content and amyl-ase activity i n this experiment, since the starch-dextrin fraction of beet root tissue Is low, then beta-amylase might be of some importance in maintaining a low starch content i n root c e l l s thus making glucose available for sucrose synthesis. If, as reported (13), starch i s syn-thesized i n beet root cells only when the sucrose eon* 1 centration becomes high, then a high concentration of sucrose might be associated with a low beta-amylase ac t i v i t y . The reverse of this could also be acceptable. Tyson (8) found that catalase activity i n beet leaves i s positively correlated with vigor and growth i n sugar beet plants. Metabolically active plant tissues usually have a high rate of respiration and a correlated high enzymatic act i v i t y . Measurements of the catalase activity of a tissue are therefore often used as an index of the intensity of metabolic activity i n that tissue. The apparent activity of catalase should increase i f a higher level of respiration results i n the beet root as 59. a consequence of f o l i a r loss, since respiration rates i n sugar beets Is relatively higher In the growing crown than i n the older region of the beet, a decrease in catalase activity might be expected i n the mature cells of the root. As the respiration rate in the maturing tissues decreases with age, the catalase "activity index" becomes lower. A higher catalase activity might be expected i n the beet crown follow-ing f o l i a r loss and active leaf regrowth. The tissue used for catalase determination was sampled from the diagonal sections of the beets and the crown portion of the root was not included. An increase i n activity was found for a l l treatments when the T/C values are used as the basis of activity deter-mination* The increase occurred from the 1st to the 8th day. When the actual monomolecular values for each day after treat-ment are compared, i t i s seen that catalase activity i n each group decreases as follows: from the 1st to the 16th day i n the frozen beets; from the 1st to the 11th day i n the de-foliated and continually defoliated beets; from the 1st to the 8th day i n the decrowned and control beets* The sudden increase i n catalase activity, based on T/C values might indicate a higher level of respiration i n the mature beet root cells, the energy of respiration used i n the movement of soluble substances through the c e l l membrane* Based on actual monomolecular values, catalase activity 60. decreases with time i n the older ce l l s of the beet root following f o l i a r loss* prom the results of the second experiment, a few correlations can be made between invertase activity and sucrose content, invertase activity Increased over the con-trols i n both the f o l i a r frozen and decrowned beets following treatment. The highest sucrose loss based on dry weight occurred in the decrowned beets, suggesting a correlation, as proposed by oparin (7), between sucrose content and invert-ase activity* A positive correlation between invertase action and sucrose content i s not evident In both groups of defoliated beets. It i s well known now that the enzyme invertase i s actually present i n beet root tissue, but i t s role in sucrose metabolism i s unknown. From the results of this experiment, It seems unlikely that invertase i s alone responsible for sucrose disappearance, although i t i s most probably a factor* 10. SUMMARY S.K.S-R-11 sugar beet seed obtained from the B.C. Sugar Go. Ltd., Vancouver, B.C., was germinated i n January, 1954. The seedlings were transplanted to f l a t s i n March 1954 - 60 plants per f l a t * The young beets were transplanted to the f i e l d i n May, 1954* 6f % The beets were spaced approximately 8 Inches and the rows 1% feet apart* Seven rows of the best appearing beets were selected and 30 groups consisting of 10 beets per group were chosen at random from the seven rows of plants* pour different treatments and a control were used* The treatments consisted oft f o l i a r freezing; a single defoliation; continual de-foliation; decrowning* six groups of beets, or a total of 60 beets, received one type of treatment. This was repeated for each treatment giving a total of 300 beets used Including the controls. Freezing was accomplished using dry (C02) ice, and defoliation by knife removal of the leaves approximately £ inch above the beet crown. The decrowned roots were sliced immediately below the position of the lowest merlstematlo bud. Two experiments were set up, the f i r s t i n August, 1954, when the beets were harvested 1, 4, 8, 11, 16 and 20 days after f o l i a r removal; the second i n October, 1954, the beets being harvested 1, 4, 8, 12 and 15 days following treatment. Enzymes only were determined i n the second ex-periment* Ten beets from each treatment constituted a "sample"* The beets were removed from the s o i l , any foliage removed and the beet crowns sliced off. The beets were thoroughly washed < 5 2 . and a uniform diagonal section cut from each beet. The sections were pulped i n a meat grinder and the pulp well mixed* The removed leaves were weighed along with the roots harvested* Analyses were carried out on fresh beet pulp for catalase. phosphatase, phosphorylase,, invertase, beta-amylase, sucrose, Invert sugars and dry weight, some of the fresh beet pulp was stored i n a -10°c refrigerator for future analyses. In the second experiment, the diagonal section of the beet root was used for beta-amylase and phosphorylase determination, while a crown slic e was employed i n the estimation of invertase activity. Total nitrogen determinations were done on 1 gram weights of the original dried pulp. Ten grams of dried pulp were extracted with 70 percent ethanol. The alcohol extract was analysed for sucrose and invert sugars, insoluble nitro-gen and 81arch-dextrin estimations were made on portions of the alcohol extracted pulp. The validi t y of the sampling method was tested by pulping the tissue of five beet roots, mixing i t well, draw-ing five separate 40 gram aliquots of the pulp, and deter-mining the phosphatase activity present i n each, sucrose was also analyzed for using duplicate 26 gram aliquots of 63. fresh pulp. The percent dry weight decreased In a l l treated beets from the 1st to the 20th days after treatment, percent dry weight showed l i t t l e change i n the control beets. In a l l treatments, the amount of total nitrogen de-creased from the 1st to the 8th day and then increased after this time. Insoluble nitrogen decreased from the 1st to the 8th day in the decrowned and defoliated beets. Insoluble ni t r o -gen results were generally inconclusive, soluble nitrogen decreased In a l l oases except the defoliated beets to the 8th day and then Increased. The percent of sucrose, based on dry and fresh weights decreased following a l l treatments. The continually defoliated and decrowned beets lost the highest dry weight percentages of sucrose. The average fresh and dry weight percent of sucrose in the control beets was higher than the average percents in the treated groups. The results for invert sugars indicated an increase i n the amount of reducing sugars following f o l i a r loss. The percent of Invert sugars i n the control beets remained f a i r l y constant. The highest rise in inverts was found i n the decrowned beets, with the least rise in the continually defoliated beets. The percent of starch-dextrins tended to decrease i n a l l treatments. 64* A definite decrease i n percent of total carbohydrates occurred i n a l l treatments* The highest aotual value noted was i n the oontlnously defoliated beets, while the decrowned beets had the lowest value* Total carbohydrate content i n the control beets remained constant. The apparent activity of phosphorylase decreased with time i n a l l treatments. Activity In the control beets from the 11th day was considerably higher than i n the treated beets. phosphatase activity deoreased from the 1st to the . 11th day i n every treatment except deorowned. .The activity of tills enzyme increased between the 11th and 20th days. In the decrowned beets, phosphatase activity increased slightly towards the 4th day. then decreased u n t i l the 16th day. Beta-amylase activity was detected, but there were no apparent changes i n activity. There was a tendency for activity i n the treated beets to be lower than that of the controls i n the f i r s t experiment, but this was not duplicated i n the second experiment. Catalase activity, on the basis of monomolecular values, decreased with time after treatment, on the basis of T/C values, the activity increased i n the frozen beets from the 1st to the 11th day and then decreased. A continual decrease i n apparent activity was noted for the decrowned 65* beets* A positive correlation between invertase activity and sucrose loss i s suggested from the results* In experiment II, invertase activity increased i n the frozen and decrowned beets from the 1st to the 8th days* The highest sucrose losses were found i n the decrowned beets* Invertase activity i n both defoliated groups was generally lower than i n the control beets* The results found i n this experiment were largely negative• SUGGESTIONS FOR FUTURB WORK 1* Research work to determine the biochemical reactions associated with the thawing of sugar beet crown tissue* 2* Studies, of sugar beet respiration and centres of respiration* 3* Respiratory enzyme measurements* 4* Chemical analyses of various regions of the beet root in order to determine any differences in composition which might be related to sucrose loss* cork borer sections could possibly be employed* 5* Analysis of various regions of the beet root for invert sugars during sucrose loss* This might indicate the main centre or centres of sucrose disappearance* 6* Analysis of the beet crown for nitrogenous com-pounds* 7* A oloser examination of invertase activity in the beet root and i t s suggested relation to sucrose metabolism* 8* studies to determine whioh type of sampling method is most accurate - including replication of treatments and analyses* 9* studies with enzyme inhibitors in relation to the reduction of sugar losses, io^oa&tat© might be used* 10* studies involving certain growth substances, as maleic hydrazide, and their efficiency in reducing sugar losses following foliar freezing* 11• Research to find sucrose losses i n p i l e storage freezing* the exact relationship between and losses caused by f o l i a r / .68. 12. LITERATURE CITED (1) PETO, P.H. Effect of frost on sugar content In beets, proceedings of the American society of sugar Beet Tech-nologists. 108-111, 1952. (2) DOWLING, R.N. sugar Beet and Beet sugar. William Brendon and Sons Ltd. (Plymouth) 1928. (3) THE SUGAR REPINING INDUSTRY. Dominion Bureau of Stat-i s t i c s . Dept. of Trade and commerce, Canada. 1952. (4) PETO, P.H., SMITH, W.G. and LOW, F.R. Effects of pre-harvest sprays of maleic hydrazide on sugar beets* proceedings of the American Society of sugar Beet Tech-nologists. 101-107, 1952. (5) STROHMER, P., BRIEM, H. and PALLADA, 0. Experiments on the defoliation of sugar beets. Mitt. chem. Tech. Vera. Stat. cent. Ver. - Rubenz Indus. Osterr. ungar. No. 198. 1-12, 1908. (6) KOKINA, S* Relation between the number of leaves and the accumulation of dry mass and sugar in the sugar beet root. Physiol, untersuch. zuckerube. Erste Artikel Serie. ukrainischen Inst. Angew. Bot. sect, pflanzen physlol. 1934. (7) OPARIN, A.I. Storage of sugar i n the roots of beets and the significance of invertase. Biochemia 2, 135. 1934. (8) TYSON, J. influence of s o l i conditions, f e r t i l i z e r s , and l i g h t intensity on growth, chemical composition and enzymic ac t i v i t i e s of sugar beets. Michigan Agricult-ural Station Technioal Bulletin 108 p. 44 1930. (9) The Distribution of Catalase i n the Sugar Beet. Z. Zuckerlnd. Biehmen. 31. 201-17. (10) PFANKUGH, E. The phosphatase of the potato and Sugar Beet. Z. Physiol, chem. 241, 34-46. 1936. (11) PELLET, H. The presence of reducing sugars i n freshly harvested beets. Bull. Assoc. chim. sucr. Dlst. 32, 59-92. 1915. (12) COLIN, H. sucrose i n the sugar beet; i t s formation and disappearance. Rev. Gen. Botan. 28. 289-99. 1916; 29. 113-27. 1917% 69* (13) PEKLO, J. The Occurrence of starch i n sugar Beet Roots* Biedermann»s Zentr* 40, 336-387. 1912* (14) PDLTZ, L.M. Nitrogen and sugar yield i n beets* Jour* Agr. Res* (U.S.), 54. No* 79* 639*654* 1937. (15) OPARIN, A.I. Metabolism i n sugar beet roots at low temperatures* storage of beet root i n a frozen state* Compt* Rend* Acad* Sci. U.S.S.R. 2, 116*121* 1934* (16) BURMA, O.P. and MORTIMER, D.C. Division of Applied Biology, National Research Labs., Ottawa, Canada.* Biosynthesis of uridine Diphosphate Glucose i n sugar Beet leaf and i t s Possible Role i n sucrose synthesis* (17) HARRIS, F.S. The sugar Beet i n America. Rural Science Series. MaoMlllan Co. 1909* (18) EAMES, J.A., MacDANIELS, L.H. Introduction to plant Anatomy* McGraw H i l l Book Co* 313*314* 1947* (19) LAWRENCE, H.M. Taxonomy of Vascular plants* MacMillan CO. 477-478. 1951. (20) SUMNER, J.B., CHOU, T.C. and SEVER, A.T. phosphorylase of the Jack bean; i t s purification, estimation and properties* Arch* Bio chem. 26: 1-5* 1950* (21) GOTTSCHALK, R.G. Mi erode termination of acid phosphat-ase* Blochim* Biophys* Aota. 2; 582-589* 1948* (22) SUMNER, J.B. and SOMERS, G.F. Chemistry and methods of enzymes, second edition. Academic press, New York. 1947. (23) SUMNER, J.B. and SOMERS, G.F. Laboratory experiments i n biological chemistry. Academic presa Inc. pub-lishers. New York, N.Y. 39-41. 1949. (24) SUMNER, J.B. and HOWELL, S.F. A method for deter-mination of aaccharase activity, jour. B i o l . chem. 108, 51. 1935. (25) SOMOGYI, M* A method for the preparation of blood f i l t r a t e s for the determination of sugar* jour* B i o l * Chem* 86, 655 (1930); 70, 599 (1926)* (26) ANALYSIS OF SUCROSE IN BEETS* Association of O f f i c i a l Agricultural chemists* 1946* 70. (27) L00MIS. W.E. and SHULL, C.A. Methods In plant physio-logy. McGraw-Hill Book Company. New York. 1937. (28) SAILLARD, E. The reducing sugar content of the beet during i t s vegetation. Arc. Held. synd. suppl. 1658. 1921. Z. zuokerind. Czechoslov. Rep. 45, 234. 1921* (29) STEHLIK, V* Ztsehr. Zuckerindus. Czechoslovak Re pub. 49. 1-7. 1924. (30) MEYER, B.S. and ANDERSON, D.B. plant physiology. D* Van No strand Co. Inc. New York. 2nd edition. 638-639. 1952. 

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