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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 t h e s i s as conforming t o the standard r e q u i r e d  from c a n d i d a t e s  f o r the degree^of MASTER OF ARTS  Member^ o f the Department o f 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 - l l , was o b t a i n e d from the B.C. Sugar Co. L t d . , Vancouver, B.C. and germinated i n f l a t s i n a greenhouse on January 29, 1954. The beet p l a n t s were t r a n s p l a n t e d t o 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 d e s i g n was chosen i n o r d e r to reduce e r r o r caused by s o i l d i f f e r e n c e s , m o i s t u r e v a r i a t i o n s , and pH e t c . T h i r t y groups o f 10 b e e t s p e r group were s e l e c t e d from the randomized l o t . The l e a v e s o f 6 groups were f r o z e n w i t h d r y i c e and the o t h e r groups were d e f o l i a t e d , decrowned c o n t i n u o u s l y d e f o l i a t e d , o r used as c o n t r o l s . " The regrowth on the c o n t i n u o u s l y d e f o l i a t e d b e e t s was removed e v e r y two days f o l l o w i n g i n i t i a l defoliation. 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 l e a v e s o n e - q u a r t e r i n c h above the crown. Decrowning was done by c u t t i n g the beet r o o t t r a n s v e r s e l y j u s t beneath the o u t e r r i n g o f m e r i s t e m a t i c buds. The: d e f o l i a t e d b e e t s were used t o serve as a p a r a l l e l t o the d e s t r u c t i o n o f l e a v e s by f r e e z i n g . The c o n t i n u o u s l y d e f o l i a t e d b e e t s were a check on the d e f o l i a t e d b e e t s , 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 regrowth l e a v e s would p a r t i a l l y o f f s e t a l a r g e 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. H a i v e s t t i m e s were 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 Aug. 16, and at the 1, 4, 8, 12 and 15 day i n t e r v a l s f o l l o w i n g October 13. Enzyme a c t i v i t y o n l y was determined i n the second experiment. 'The f r e s h l e a f w e i g h t s o f the d e f o l i a t e d and c o n t r o l b e e t s were r e c o r d e d and l a t e r compared w i t h l e a f regrowth w e i g h t s and sugar c o n t e n t . The b e e t s were h a r v e s t e d i n groups o f 10 b e e t s a l l t r e a t e d i n one s p e c i f i c manner. Ten b e e t s o f each group were removed from the s o i l and each beet s l i c e d d i a g o n a l l y a c r o s s the c e n t r e r e g i o n . The s e c t i o n s were washed i n water and p u l p e d i n a meat g r i n d e r g i v i n g a p p r o x i m a t e l y 2000 grams o f p u l p from 10 s e c t i o n s . Three hundred grams o f p u l p was used i n dry weight -determination. F o r t y grams o f f r e s h p u l p from each group was blended f o r 2 minutes w i t h 100 m l . of. d i s t i l l e d i c e water i n a Waring b l e n d o r . The s o l u t i o n was f i l t e r e d through b r o a d c l o t h and used i n enzyme a c t i v i t y measurements. In the second experiment, 47 grams o f p u l p was b l e n d e d w i t h 100 m l . o f d i s t i l l e d i c e water f o r 2 m i n u t e s . The crown p o r t i o n o f the r o o t was used i n the e s t i m a t i o n o f i n v e r t a s e a c t i v i t y . A check on the sampling method showed t h a t the 40-gram a l i q u o t o f pulp used f o r enzyme d e t e r m i n a t i o n r e p r e s e n t e d the sample. Sucrose p e r c e n t and phosphatase a c t i v i t y were used as the b a s i s o f t h i s t e s t . The f r e s h p u l p was a n a l y z e d f o r s u c r o s e , i n v e r t sugars, d r y w e i g h t , c a t a l a s e , p h o s p h o r y l a s e , beta-amylase and i n v e r t a s e enzyme a c t i v i t i e s . The d r i e d p u l p was ground t o 40-mesh and a n a l y z e d f o r t o t a l n i t r o g e n , sucrose and i n v e r t s u g a r s . I n s o l u b l e n i t r o g e n and s t a r c h - d e x t r i n s were determined i n e t h a n o l e x t r a c t e d p u l p . D u p l i c a t e d e t e r m i n a t i o n s were made on each sample. P e r c e n t a g e s are based on both d r y and f r e s h w e i g h t s and g i v e n as T/C v a l u e s .  P h o s p h o r y l a s e , phosphatase, c a t a l a s e , beta-amylase, i n v e r t a s e were measured. Sucrose, i n v e r t sugars, s t a r c h - d e x t r i n 3 , and t o t a l and i n s o l u b l e n i t r o g e n were a l s o determined. T h e . h i g h e s t amount o f l e a f regrowth o c c u r r e d 4 - 1 7 days a f t e r f r e e z i n g . 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 w e i g h t s and sucrose content nor between r o o t weight and sugar content i n mature b e e t s . The p e r c e n t • d r y w e i g h t • d e c r e a s e d i n a l l t r e a t e d b e e t s from the 1 s t to the 20th days a f t e r t r e a t m e n t . T h i s decrease i s l i k e l y a r e s u l t o f sucrose l o s s and an i n c r e a s e d h y d r a t i o n i n the beet r o o t . Sucrose p e r c e n t based on d r y and f r e s h weight g e n e r a l l y d e c r e a s e d f o l l o w i n g a l l - t r e a t m e n t s . A p o s i t i v e c o r r e l a t i o n between p e r c e n t sugar l o s s and l e a f regrowth i s suggested. There was an i n c r e a s e i n the amount o f r e d u c i n g sugars a f t e r f o l i a r l o s s . The s u g g e s t i o n has been made t h a t the monosaccharide sugars are u t i l i z e d almost immediately i n l e a f regrowth o r i n (increased r e s p i r a t i o n i n the beet crown. The percent, o f s t a r c h - d e x t r i n s tended t o decrease i n the t r e a t e d b e e t s 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 p e r c e n t o f t o t a l c a r b o h y d r a t e s found f o l l o w s the f a c t t h a t sucrose d i s a p p e a r s . T o t a l carbohydrate e s t i m a t i o n s seem to p r o v i d e a r e a s o n a b l e b a s i s f o r d e t e r m i n i n g the amount o f sucrose los's. T o t a l and s o l u b l e n i t r o g e n v a l u e s decreased t o the 8 t h day a f t e r treatment and i n c r e a s e d a f t e r t h i s t i m e . I n s o l u b l e n i t r o g e n r e s u l t s were g e n e r a l l y i n c o n c l u s i v e . The r e s u l t s suggested a t r a n s l o c a t i o n o f s o l u b l e n i t r o g e n o u s compounds to the beet crown where a c t i v e growth was occurring. The apparent a c t i v i t y o f p h o s p h o r y l a s e decreased w i t h time i n a l l t r e a t m e n t s . S t a r c h phosphorylase i n sugar beet r o o t l i k e l y has a minor r o l e i n t o t a l carbohydrate metabolism o f the t i s s u e . Phosphatase a c t i v i t y decreased t o -the 11th day i n e v e r y treatment except decrowned. The r e a s o n f o r a lower apparent phosphatase a c t i v i t y i n t r e a t e d beets i n t h i s experiment i s not known. I t may be a s s o c i a t e d w i t h an i n c r e a s e 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 c o u l d be found between s t a r c h - d e x t r i n content and amylase activity. C a t a l a s e a c t i v i t y based on monomolecular v a l u e s , decreased w i t h time a f t e r t r e a t m e n t . A decrease i n c a t a l a s e a c t i v i t y might be expected i n the mature, c e l l s o f the r o o t as the r e s p i r a t i o n r a t e d e c r e a s e s w i t h age, A c o r r e l a t i o n between i n v e r t a s e a c t i v i t y and sucrose l o s s was i n d i c a t e d i n the f r o z e n and decrowned b e e t s 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 o f t h i s experiment i t seems u n l i k e l y t h a t i n v e r t a s e i s alone r e s p o n s i b l e f o r a sucrose d e c r e a s e . The r e s u l t s found i n t h i s experiment were l a r g e l y n e g a t i v e .  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  the concern o f Canadian  sugar b e e t t e c h n o l o g i s t s over  from  sugar  l o s s i n c u r r e d by e a r l y f a l l f r o s t s b e f o r e and d u r i n g sugar b e e t h a r v e s t i n southern A l b e r t a , Canada*  This p a r t i c u l a r  phase o f sugar b e e t p h y s i o l o g y r e s e a r c h stemmed from a con* ference on p h y s i o l o g i c a l r e s e a r c h problems I n the sugar b e e t i n d u s t r y , h e l d a t the Dominion E x p e r i m e n t a l s t a t i o n , L e t h b r i d g e . A l b e r t a on May 8, 1952*  A t t h i s conference i t was  r e s o l v e d t h a t a r e s e a r c h program be i n i t i a t e d f o r the study o f b a s i c p l a n t p h y s i o l o g i c a l problems i n c o n n e c t i o n w i t h the p r o d u c t i o n o f sugar b e e t s *  I t was a l s o r e s o l v e d t h a t such a  program should i n c l u d e r e s e a r c h c o n c e r n i n g the e f f e c t s o f f r o s t on growth and sugar content o f sugar b e e t s *  sugar  l o s s e s d u r i n g p i l e storage o f b e e t s were d i s c u s s e d a t the conference* I t has been known f o r some time t h a t one e f f e c t o f autumn f r o s t s on sugar be,©ts i s the d e c l i n e i n growth and sucrose content; o f the b e e t r o o t .  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 r i s e u n t i l harvest time unless a 26°p f r o s t ooeurs*  The sugar content i s usually decreased  following the f i r s t f r o s t , regardless of i t s i n t e n s i t y below 26°p.  AS t h i s decrease was found to appear within f i v e days,  i t was not associated with obvious l e a f regrowtiuU). The percent sucrose reduction i s quite serious i f a warm period follows the f i r s t f r o s t *  Good evidence e x i s t s  that early f r o s t s are responsible f o r low sugar content i s given by the f a c t that i n 1949 i n Alberta, a k i l l i n g f r o s t occurred on September 12 and the average percent of sugar on September 30 was only 14*5 percent*  I t i s believed that t h i s  early f r o s t was responsible f o r the abnormally low average sugar percent of 14*5 at the 30 day point* based on a 20 years average*  These r e s u l t s are  S t a t i s t i c a l methods have shown  a p o s i t i v e c o r r e l a t i o n between f r o s t and average sugar percent of harvested beet roots (1)* Late second growth of tops causes a reduction i n sugar content*  Where early f r o s t s are followed by warmer  periods, second growth i s favoured*  sucrose l o s s maynot  occur when second growth i s absent and the i n i t i a l f r o s t period p e r s i s t s , keeping the a i r and s o i l temperature low (2)* Storing sugar beets i s a common p r a c t i s e and the question of l o s s of sugar and weight during storage has re-  3.  ceived c a r e f u l attention,  sugar l o s s r e s u l t s from r e s p i r a t i o n  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* of -1*7°C (2)*  This means a  temperature  Respiration i s accompanied by a change of the  sucrose into the i n v e r t sugars glucose and fructose*  The  sucrose l o s s i s greater i n beets which have not been decrowned* Under favourable conditions of temperature This w i l l cause a sucrose reduction*  "sprouting" occurs*  Temperature i s  important i n the degree of l o s s , that i s , the higher the temperature  the more active are the changes*  I f a suitable  temperature  f o r sugar conversion and r e s p i r a t i o n be  accompanied by drought, then the l o s s i s high (2)*  Thus,  l e a f regrowth during p i l e storage and the r e s u l t i n g sucrose reduction may be a problem which i s s i m i l a r l y , I f not d i r e c t l y , related to sucrose l o s s and l e a f regrowth following autumn frosts* The problem of sucrose disappearance following frosts i s one of economic importance  since the l o s s of sugar i n the t  beet root may be aa highaas l . l percent ten days a f t e r f r o s t (1)*  The concern o f the p l a n t biochemist i s i n how  these  f r o s t s i n i t i a t e metabolic changes i n the root which r e s u l t i n the l o s s of sucrose and also, what metabolic changes are i n volved,  i t i s the purpose of t h i s experiment to discover  some o f tho metabolic changes occurring i n the beet root following f o l i a r l o s s and subsequent l e a f regrowth. The sugar beet industry i n Canada has shown steady growth since 1881.  The most recent figures that could be  obtained f o r the Canadian sugar industry show that i n 1952 the t o t a l y i e l d of beets was 1,022,693 tons, harvested from 92,607 acres and valued at 12,681,393 d o l l a r s (3).  i n 1950,  the  three Alberta f a c t o r i e s located i n 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 l o s s of 1.0 percent sucrose would be equivalent to 700,000 pounds or 350 tons of manufactured beet sugar. Studies of the s p e c i f i o reasons f o r sucrose l o s s i n beet roots which have had t h e i r foliage removed are few. L i t t l e e f f o r t i n the past has been given to the biochemical aspects of the problem,  some related physiological and b i o -  chemical aspects are outlined i n the following l i t e r a t u r e review: A study of twenty years harvest results and average sugar content f o r the Canadian sugar Factories at Raymond, Alberta, have shown, that k i l l i n g f r o s t s of 26°p. p r i o r 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 d e f o l i a t i o n o f sugar beets by F* strohmer e t a l (5)., the conclusion i s t h i t complete as well as p a r t i a l d e f o l i a t i o n of the sugar beet reduces both the t o t a l y i e l d o f sucrose and the t o t a l weight of the beets* A r e l a t i o n s h i p between the, number o f leaves and the accumulation o f dry mass and sugar i n beetaroots has been noted (6). Removal o f more than h a l f the leaves (2-4 l e f t ) caused a pronounced decline i n the y i e l d of dry matter of the root but only a s l i g h t decrease i n the percent sugar per plant* Increase i n p r o d u c t i v i t y 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 o f  photosynthate might remain where synthesized and hinder the assimilating process i n the root* Enzyme studies using beet root tissue have shown the following: A*I* Oparln (7), i n h i s studies of beet root i n v e r t ase, succeeded i n establishing the presence o f the enzyme i n sugar beet root*  I t was found possible to reveal h y d r o l y t i c  a c t i v i t y i n suspensions prepared by maceration o f the top parts o f the root, but i n the central portion of the root, i n vertase a c t i v i t y was apparently absent*  The following  relationships between invertase a c t i v i t y and sugars fere found:  6. 1. Tne v a r i e t y with the maximum Invertase  activity  has the lowest t o t a l sugars and percent dry material. 2. An invertase a c t i v i t y increase i s accompanied by lower proportional sucrose and higher proportional monosaccharide i n beet roots. 3. An inverse relationship e x i s t s between invertase a c t i v i t y and the r a t i o o f sucrose to monosaccharide. Oparin believes that invertase a c t i v i t y 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 r e s u l t o f i n vertase synthetic action.  The exact r o l e o f Invertase i n  sugar beet root i s unknown. J . Tyson (8) found that catalase a c t i v i t y i n beet leaves i s p o s i t i v e l y correlated with vigor o f growth and size of sugar beet plants.  Catalases are unevenly d i s t r i b u t e d i n  the beet root, being more aetive near the epidermis and decreasing towards the centre o f 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,  that phosphatase serves as a "brake  11  i t i s suggested  on the degradation of  carbohydrate by s p l i t t i n g o f f 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 o f the sugars present i n normal beet roots*  These studies have  concerned synthesis, translocation, and r e l a t i o n 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 o f the beet root; r a f f i n o s e about 0.6 to 1.0 percent; glucose and fructose 1.0 to 2.0 percent; arabinose i n traces (2). prom studies o f the presence o f reducing sugars i n freshly harvested beets by H. p e l l e t (11), the conclusions made were: 1. Freshly cut beets contain from 0*04 to 0*10 percent reducing sugars based on fresh weight and most contain from 0.08 to 0.05 percent* 2. There i s no r e l a t i o n between the percent of sucrose and the percent o f reducing sugars. 3. The amount of reducing sugars i s p r a c t i c a l l y constant throughout growth. From researoh o f other workers (12) interested i n sugar beet carbohydrates and t h e i r metabolism, i t has been  8*  noted that: 1. The percentage of fructose Increases as the growing season progresses.  A low l e v e l of fructose i s associated  with rapid growth and a higher l e v e l with slow growth and maturity. 2. Fructose, glucose, and sucrose a l l show d i u r n a l variations within the l e a f 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 p o l a r d i r e c t i o n . 4. The proportion of reducing sugars Immediately above the crown of the root i s 50 percent of the t o t a l 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 i n the c e l l s of the sugar beet root i s connected with the presence o f leucoplasts i n the c e l l s . Starch synthesis can r e a d i l y he noticed i n roots with a high percent of sugar. The r e l a t i o n of nitrogen to accompanying changes i n composition of beet roots has been followed (14). The u t 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 p l a n t .  9* Studies of the metabolism i n sugar beet root at low temperatures (15) have shown that below - 2 ° c ,  respiration  with l o s s 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 i n 1 hour. tinued 4 days.  Respiration at -12°C con-  On thawing, i t was found that sucrose i s con-  verted into glucose and fructose.  Through complete freezing  by severe natural f r o s t , l o s s of sucrose i n the beet root i s reduoed more than 35 percent i n r e l a t i o n to sucrose l o s s caused by p a r t i a l 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 i n v e s t l g a t i r s found that when r a d i o -  active glucose -1 -phosphate  (G-l-P) i s fed to an excised sugar  beet l e a f through i t s p e t i o l e , radioactive sucrose, equally l a b e l l e d 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) y i e l d s radioactive TJDPG i n the l e a f homogenates.  The enzymatic  a c t i v i t y i s associated with the c e l l sap cytoplasm.  G-l-P  can not be replaced by free glucose, fructose or sucrose f o r 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 e i t h e r free fructose or fructose 1-6 diphosphate f o r the biosynthesis o f sucrose, that the Intermediates  i t i s possible  i n sucrose synthesis i n the beet l e a f  are afctive i n beet root -tissue. Such substances as oxalate, asparagine, glutamine, a l l a n t o i n and betain can be detected i n considerable concentration i n sugar beet roots. 2.  THE SUGAR BEET PLANT The sugar beet belongs to the goosefoot family, or  Ghenopodiaceae.  The c h i e f c u l t i v a t e d members o f t h i s family  are beets and spinach.  The species Beta v u l g a r i s  includes  sugar beets, mangel wurzels, common garden beets, and l e a f beets.  There i s a wild form o f the same genus (Beta maritima)  which grows as a perennial along the coast o f northern Europe* The sugar beet i s o r d i n a r i l y a b i e n n i a l , storing sucrose i n the root during the f i r s t year, and sending up seed stalks i n the second. most o f the leaves.  They bear the flowers, seeds, and  The stem, i n the f i r s t year, consists of  the crown on top o f the root.  The crown i s very much short-  ened and scarcely distinguishable from the f l e s h y root. A  The enlarged fleshy taproot i s the predominate part of the beet p l a n t .  A cross o r transverse section o f the root  w i l l show a number o f concentric rings, and between each there i s a s o f t , thin-walled c e l l u l a r material or storage  11. parenchymatous tissue containing the c e l l sap.  The coneentric  rings are made up of c o l l a t e r a l fibro-vascular bundles comprising the phloem, the cambium, and the xylem.  The  phloem i s composed of sieve tubes, companion c e l l s , and t h i n walled meristematic c e l l s of rectangular shape, which are joined with s i m i l a r c e l l s a r i s i n g i n the medullary rays, thus g i v i n g the appearance i n 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 t h e i r early stages of development, but t h i s disappears and secondary thickenings appear on the c e l l w a l l s .  The vessels serve i n water and dissolved  mineral transport (17). The formation of secondary cambial zones i s responsible f o r 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 i r r e g u l a r l y arranged bundles.  These bundles are p a r t l y of a  secondary nature, but camblal a c t i v i t y soon ceases and a  new,  secondary cambium arises i n the pericycle j u s t outside the bundles.  The a c t i v i t y 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, t r a n s i t i o n region, and several internodes of stem.  The f i r s t cambium forms a r i n g of bundles close  about the primary xylem.  A secondary cambium arises i n the  12. pericycle and t h i s i s followed i n rapid succession by others originating similarly.  A l l layers continue to function,  perhaps i n d e f i n i t e l y * though more slowly a f t e r an e a r l y period of a c t i v i t y *  The cambium a r i s e s apparently as a  continous  band, but forms more or l e s s separate bundles with bands of conjunctive parenchyma developing between the vascular s t r i p s * The p o s i t i o n of each new  cambium, as i t arises i n the p e r i c y c l e ,  i s such that i t encloses a few layers o f p e r i c y e l i c c e l l s * These multiply and b u i l d a parenchymatous l a y e r as r a p i d l y as the cambium builds the vascular l a y e r *  Alternate bands of  p r o l i f e r a t e d p e r i c y c l e and of vascular bundles are then foisned. The former constitute the dark-colored, colored rings i n the beet root*  the l a t t e r the l i g h t -  The bundles are l a r g e l y  parenchymatous with only a few l i g n i f i e d c e l l s i n the xylem. Growth continues In the bundles apparently both by oambial a c t i v i t y and by p r o l i f e r a t i o n 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 I r r e g u l a r l y 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. f i v e connate sepals*  The perianth i s u n i s e r i a t e , consisting of Petals are absent*  There are f i v e  stamens opposite the calyx lobes and p a r t l y attached to the perianth ring*  The filaments are d i s t i n c t and the anthers  2-celled, dehiscing l o n g i t u d i n a l l y .  There Is one p i s t i l  and  13. the i n f e r i o r ovary i s p a r t i a l l y embedded i n the f l e s h of the receptacle.  The ovary i s 1-loculed, 1-ovuled, 3-carpelled.  The f r u i t i s an indehlscent n u t l e t .  The flowers are produced  i n dense cymes along an axis, r e s u l t i n g 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 l a t e r a l s a r i s e (19).  13.  MATERIALS AMD METHODS  -  CULTIVATION OF THE SUGAR BEETS FOR  THE EXPERIMENT  S.K.E-R-ll sugar beet seed~obtained from the Sugar Co. L t d . , Vancouver, B.C., January 29, 1 9 5 4 * on March 25  was  germinated i n f l a t s  The s e e d l i n g s were thinned and  to other f l a t s .  B.C. on  transplanted  Approximately s i x t y sugar b e e t  s e e d l i n g s were p l a c e d i n each f l a t : and 15  flats  prepared.  The p l a n t s were k e p t under moderate l i g h t c o n d i t i o n s i n a greenhouse May  2,  from where they were t r a n s p l a n t e d to the f i e l d  1954.  The b e e t r o o t s were, on the average, 3 i n c h e s The young p l a n t s were spaced 8 to 12  i n length. the  field  900  beets were p l a n t e d c o n s i s t i n g of 15  p e r row.  and the rows p l a n t e d l£ f e e t a p a r t .  inches i n  Approximately  rows w i t h 60 beets  The s o i l c o n d i t i o n a t the time of p l a n t i n g was  f a v o u r a b l e f o r beet r o o t s .  I t was m a i n l y a sandy loam  had become packed a t the 6 i n c h below s u r f a c e l e v e l . fertilizer for  content of the s o i l was  proper b e e t r o o t growth and  added r M ^ & ^ - s r p l a n t i n g .  c o n s i d e r e d as commercial  not  which The  being^adequate  fertilizers  The beetB were w e l l watered d u r i n g  the  summer months by a s p r i n k l e r .  the  p e r i o d from May  The p r e c i p i t a t i o n d u r i n g  to August, 1954» was  days were o v e r c a s t and d u l l . as the s o i l  on  q u i t e heavy and many  ThiB c l i m a t i c c o n d i t i o n as w e l l  c o n d i t i o n would tend to favour a low sugar c o n t e n t .  The b e e t s , a t h a r v e s t time i n August 1954» had not reached commercial m a t u r i t y and were c o n t i n u i n g to s t o r e sugar i n the root.  The average r o o t s i z e was  3<*4 inches across the c e n t r e  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 were selected.  appearanoe  Lots of ten beete were chosen from each row,  giving a total number of 30 l o t s amongst the 7 rows.  Each  8 i n g l e l o t was given a number and then the numbers were randomly picked.  Diagram I I shows the arrangement of the p l o t .  This randomized l o t design was chosen i n order to reduce errors caused by s o i l differences, moisture v a r i a t i o n s , pH, and s o i l structure e t c .  Since the total area of the beet p l o t  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 d e f o l i a t e d , decrowned, continously d e f o l i a t e d , 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) i c e was placed i n a rack i n 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  10  5  4 9  4  I  14  18  22  26 i  i 8  13  17  21  i  t  25  29  i  I 12  6  11  16  20  15  19  S  24  23  28  27  DIAGRAM I I - 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  *  26  30  27  20  28  9  6  i£.  .  merits made during the freezing period gave readings of -16° C. at 2 inches above ground l e v e l ami-Ae,'. the box, and an average a i r temperature within the box of - 1 1 ° C , 2 inches below the dry i c e . The outside a i r 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 p a r t i a l l y exposed to f r e e z i n g while others were beneath the s o i l surface.  No attempt was  made to protect the crown as i t i s usually exposed i n f i e l d beets grown commercially*  Also, i t was desired to duplicate  fi.«)Wx conditions as c l o s e l y as p o s s i b l e .  The weather was  humid and overcast on August 16, 1954, the day of freezing* One hundred and twenty of the beets were d e f o l i a t e d with a k n i f e by s l i c i n g o f f 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. defoliation*  Sixty of these beets were not treated following The remaining d e f o l i a t e d beets were p e r i o d i c a l l y  redefoliated every two days. Sixty beets were decrowned.  Decrowning was  accomplished by cutting the beet root transversely just beneath the outer ring of meristematic buds.  The out surfaces  were l i b e r a l l y coated with p a r a f f i n wax, this being done to prevent fungous and b a c t e r i a l attack. The defoliated beets were used to serve as a p a r a l l e l to the destruction of leaves by f r e e z i n g .  The continually  DIAGRAM I I I - GUT-AWAY END VIEW OP BOX USED TO FREEZE BEETS IN THE FIELD  d e f o l i a t e d b e e t s s e r v e d as a c h e c k where i t was  root*  root  No  as l o n g  partially  offBet  p h o t o s y n t h a t e would as  beets re-  a l a r g e sugar l o s s i n  be  translocated  t h e l e a v e s were r e m o v e d , h e n c e  c a r b o h y d r a t e changes tinually  the d e f o l i a t e d  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 t h e new  g r o w t h l e a v e s would the  on  to the b e e t  different  were e x p e c t e d i n t h e r o o t s o f t h e  defoliated beets.  Beets w i t h t h e i r  con-  crowns removed  were u s e d i n o r d e r t o d e t e r m i n e w h e t h e r s u g a r c h a n g e s  would  o c c u r as r a p i d l y  had  when t h e m e r i s t e m a t i c c e n t r e (crown)  been d e s t r o y e d .  The f i r s t  l6, to  1954«,  experiment  t h r e e and one  the f i e l d .  At various  time i n t e r v a l s  b e e t s a l l t r e a t e d i n one  h a r v e s t e d i n the f i r s t 20  day i n t e r v a l s The  October  this  experiment.  first  experiment  and  w i t h h a r v e s t s on  treatments. The  transplanting t r e a t m e n t , the  A group  a t t h e 1,  August  consisted  of  G r o u p s were 4,  8,  11,  16,  and  16.  t r e a t m e n t commenced the  1, 4, 8, 12,  on  and  15th  Enzymes o n l y were d e t e r m i n e d i n  b.e.et^rtqpips u s e d were s i m i l a r  as i n the  experiment* The  weighed the  ten,  f o l l o w i n g August  13, 1954,,  following  of  from  s p e c i f i c manner.  second experiment  days  t r e a t m e n t b e g a n on  h a l f months f o l l o w i n g  b e e t s were h a r v e s t e d i n g r o u p s 10  and  topB  of the d e f o l i a t e d  when r e m o v e d ( a t  <UfeJ»*tij4 b e e t s , and  and  the b e g i n n i n g o f  control beets  were  the e x p e r i m e n t f o r  a t each h a r v e s t f o r the c o n t r o l s .  The  freBh leaf recorded sugar  weights  and l a t e r  content.  l e a v e s was topped  of  the d e f o l i a t e d  and  compared w i t h l e a f  The  average  control  regrowth  f r e s h weight  beets  weightB  of the  regrowth  Leaf regrowth  weights  o b v i o u s l y n o t be m e a s u r e d i n t h e c a s e o f t h e c o n t r o l  PROCEDURE USED I N THE  PREPARATION OP  The b e e t r o o t s u s e d  cone.  type.  i n this  Sugar grooves  were a b s e n t tips  Pew  of  p e n e t r a t e d d i r e c t l y downwards i n t o  soil.  c o n d i t i o n was  Secondary  soil  The  10  was  as  l e a v e s were s l i c e d  knife slices of  curled  T h i s growth-  also  the  inhibited.  follows:  o f f the f r o z e n  and  were p u l l e d f r o m The  the new  defoliated beets.  the c o n t r o l b e e t s .  were made a p p r o x i m a t e l y  t h e c r o w n r e g i o n where new  even  the r o o t s o b s e r v e d  the s e c o n d a r y r o o t s .  l e a v e s were a l s o removed f r o m  typical  t h e compact s t r u c t u r e o f  b e e t s o f each group  l e a v i n g behind most of  the  the  and  t w i s t e d and  the s o i l .  r o o t b r a n c h i n g was  The p r o c e d u r e 1.  caused by  groups.  were o f  the s i n g l e  were t i g h t l y  the bottom o f the b e e t .  limiting  experiment  and  could  BEET SAMPLES  They were g l o b u l a r i n s h a p e and n o t  t r i p l e primary root about  and  o b t a i n e d f o r t h e d e f o l i a t e d b e e t s when they, were  a t each h a r v e s t p e r i o d .  "fangy"  were  The  Transverse  i n c h below the c e n t r e  l e a v e s appear.  Any  severely  t w i s t e d r o o t s were r e m o v e d . 2.  E a c h b e e t was  r e g i o n . ( s e e diagram position).  T h i s was  sliced  I of beet  diagonally across i t s centre  t i s s u e r e g i o n s and  sugar  com-  done i n o r d e r t o a c q u i r e t h a t p o r t i o n  of  PlftfeftflM  -  SHOWING  piFPe^erMr  IN _  I  A  N  ^  Y  S  '  ^  6  V  SOC R O S E :  «E&ION«S H  .  8 > P U £  T H E tA  -  6EE.T  OlSrRIBorjON SU&-AR  CrROvJER'S  Beer A  N  N  ROOT -  U  A  L  l<\0&.  the beet where sugar concentration i s highest*  The s i z e of  the sections was approximately 2 inches i n thickness and 4 inches i n length* 3* The sections were put into a meat grinder and pulped.  This pulp was then well mixed f o r 10 minutes, i n -  eluding with i t the small amount of beet juice produced by  t  About 2000 grams of pulp was obtained from 10 beet  pulping* sections*  4* Approximately 300 grams of fresh pulp was weighed, placed i n the 110° C. oven f o r 10 minutes, removed and transf e r r e d to the 65°  0. oven where i t was dried to constant  weight (4 days).  The high temperature f o r 10 minutes i n -  activates a l l enzymes and low temperature drying avoids caramel i z a t i o n of the  sugarB.  The material was ground to 40 mesh  i n an e l e c t r i c pulverizer and stored over calcium chloride i n dessicators. .  5. Forty grams of f r e s h pulp from each group  was  weighed to the nearest tenth of a gram and blended i n a Waring blendor with 100 mis. of d i s t i l l e d i c e water f o r 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 a c t i v i t y estimations. The remaining pulp was heated to 110° C. f o r 10  6.  minutes, transferred to cpl^s+tfc-aie bags and stored i n a -10° C r e f r i g e r a t o r f o r future analyses.  1  *.  The beet roots harvested i n the second experiment i n October were treated i n the same manner and used f o r enzyme I-  ReFRItERflTAON PBPT-  OF  A  N  I  M  O A  L  B  T  A  I  N  E  D  T H f o u t H  rtuSBANORV.  THfc vi.  3 .  C.  K  I  N  D  N  E  S  S  o.F.  DR.  W o o O •.  r u n e . F o r t y seven grams of pulp was "blended w i t h 100 m i s , o f d i s t i l l e d i c e water f o r two minutes.  Diagonal s e c t i o n s of the  beet r o o t were pulped f o r the beta-amylase and p h o s p h o r y l a s e d e t e r m i n a t i o n s while •§• i n c h of the crown p o r t i o n was used i n the e s t i m a t i o n of i n v e r t a s e a c t i v i t y .  An experiment  to d e t e r -  mine the r e g i o n o f beet r o o t having the g r e a t e s t i n v e r t a s e a c t i v i t y was c a r r i e d o u t .  The r e s u l t s are g i v e n i n Table I .  VALIDITY OF THE SAMPLING METHOD On c o n s i d e r a t i o n of the sampling t h i s experiment,  i t was  thought  technique used i n  t h a t a check on the method  would show i f 40-gram p o r t i o n s o f beet pulp blended f o r enzyme a c t i v i t y e s t i m a t i o n s a c t u a l l y r e p r e s e n t e d the sample. The f i v e average  t e s t f o r v a l i d i t y was accomplished  s i z e b e e t s , c l e a n i n g , s l i c i n g and p u l p i n g them i n  a meat g r i n d e r . for  by h a r v e s t i n g  10 minutes,  The t o t a l volume of pulp was thoroughly mixed and 5*  40-gram a l i q u o t s weighed o u t .  These  pulp a l i q u o t s were blended w i t h 100 m i s . of d i s t i l l e d i c e water as q u i c k l y as p o s s i b l e and used immediately t i m a t i o n of phosphatase a c t i v i t y . fresh, pulp were a l s o made.  Sucrose  f o r the es-  analyses on the  The r e s u l t s a r e g i v e n i n T a b l e I I .  SUBSTANCES ANALYZED FOR; Analyses were c a r r i e d o u t f o r the f o l l o w i n g : F r e s h pulp  D r i e d , ground pulp  Catalase Phosphatase  Total nitrogen Sucrose  A l c o h o l extracted pulp Insoluble nitrogen  8,1.  Fresh pulp Phosphorylase Invertase beta-amylase Dry weight sucrose i n v e r t sugars  pried, ground pulp invert sugars  Alcohol extracted pulp starch-dextrins  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 equipment available i n the laboratory. A Warburg respirometer f o r respiratory enzyme a c t i v i t y measurement was not standard equipment i n the laboratory at the time of t h i s experiment* Catalase a c t i v i t y i s i n d i c a t i v e of general c e l l metabolism and plant growth (8).  phosphatase  i s Important i n  the g l y c o l y t i c scheme and the control of carbohydrate metabolism i n sugar beets (10)* metabolism:  phosphorylase i s active i n starch  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 a c t i v i t y has been correlated with sucrose content o f beet roots by some investigators  (7).  TABLE I INVERTASE ACTIVITY IN VARIOUS REGIONS OP THE BEET ROOT Section o f beet  Invertase a c t i v i t y expressed as mgms o f invert sugar X 1000  Grown  300  Beet centre  290  Outer surface  270  Beet t i p  210  TABLE I I VALIDITY OF THE SAMPLING METHOD Pulp Aliquot  phosphatase a c t i v i t y as ugms o f phenol  percent sucrose fresh weight  I  492  15.1  II  487  15.0  III  501  15.3  IV  496  15.0  V  485  14.9  zz. In a l l analyses, d u p l i c a t e determinations o f each sample., weights.  were made  P e r c e n t a g e s are based on b o t h dry and  The v a l u e T/C  fresh  i s o b t a i n e d by d i v i d i n g the v a l u e f o r  s u b s t a n c e o r a c t i v i t y i n the t r e a t e d p l a n t s by the v a l u e  ob-  t a i n e d f o r t h a t s u b s t a n c e 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.  I n the t a b l e s , the v a l u e i s t a k e n to  the u n i t p l a c e . 14'.  ANALYTICAL METHODS - FRESH PULP ENZYMES PHOSPHORYLASE - The a c t i v i t y o f t h i s enzyme  measured by the method o f Sumner e t a l , (26).  One m l . o f  zyme e x t r a c t i s i n c u b a t e d w i t h 2 m i s . of b u f f e r e d s t a r c h s u b s t r a t e f o r 1 hour a t 25°  0.  phosphates. of HOI  substrate-  i n 50 m i s . o f  dist-  S h a k i n g w i t h d r y c a l c i u m o x i d e removes i n o r g a n i c The f i l t r a t e f r o m t h i s i s n e u t r a l i z e d w i t h drops  and an equal volume of pH 6.0  c i t r a t e b u f f e r added.  T h i s s u b s t r a t e i s m i x e d w i t h an eqmal volume of a 1 potato  en-  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 o f 1 gram of g l u c o s e - l - p h o s p h a t e i l l e d water.  was  percent  s t a r c h s o l u t i o n b e f o r e use i n a d e t e r m i n a t i o n .  The  b u f f e r e d s u b s t r a t e 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 m i s . of 6.66  ammonium m o l y b d a t e .  percent  i s o b t a i n e d by adding 5 m l . o f 7*5^  sulfuric  C o l o r development acid, followed  by 5 m l . o f 4 p e r c e n t f e r r o u s s u l f a t e s o l u t i o n and of d i s t i l l e d w a t e r .  The m i x i s r o t a t e d and a l l o w e d  then 10 to  ml.  stand  for  10 minutes b e f o r e 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 a d d i n g t h e s u b s t r a t e a f t e r t h e a d d i t i o n o f ammonium molybd a t e t o 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 z e r o e d to a tube o f 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 o f p h o s p h o r y l a s e i s e x p r e s s e d as m i c r o -  grams o f phosphorus formed p e r m l . o f e x t r a c t .  A standard  phosphorus c u r v e was made b y p r e p a r i n g a s e r i e s o f phosphorus s o l u t i o n s (as KH2PO4) w i t h a range o f c o n c e n t r a t i o n s between 4 and 36O micrograms phosphorus.  C o l o r development and d i l u t i o n  were c a r r i e d o u t as f o r an a c t u a l p h o s p h o r y l a s e d e t e r m i n a t i o n . PHOSPHATASE2- Phosphatase  a c t i v i t y was d e t e r m i n e d by  G o t t s c h a l k ' s m i c r o method (2f<) , where a c t i v i t y i s measured as micrograms phenol formed p e r m l . o f p l a n t e x t r a c t .  The method  c o n s i s t s o f i n c u b a t i n g 1 m l . o f t h e p l a n t (enzyme) e x t r a c t w i t h 5 m l . o f a p h e n y l - d i e o d i u m 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 s u b s t r a t e c o n s i s t s o f 1.09  p h e n y l d i s o d i u m phosphate i n 200 m l . o f pH 6.0 made t o 1 l i t r e  with d i s t i l l e d water.  grams o f citrate buffer  The amount o f l i b e r a t e d  phenol i s e s t i m a t e d b y c o l o r development u s i n g 2 m l . o f F o l i n C i o c a l t e u r e a g e n t , w h i c h i s e s s e n t i a l l y unreduced molybdic a c i d .  phospho-  F i v e m l . o f a 10 p e r c e n t sodium c a r b o n a t e  s o l u t i o n ci'Sv added, and the phosphomolybdic  acid, i n alkaline  s o l u t i o n , i s reduced b y phenol t o a m i x t u r e o f t h e l o w e r o x i d e s of molybdenum, and t h i s m i x t u r e y i e l d s t h e t u r b i d b l u e known as molybdenum b l u e .  Affter 10 m i n u t e s , the m i x i s d i l u t e d by  adding 100 m l . o f t a p w a t e r . of  A b l a n k i s r u n by adding 1 m l .  t h e enzyme e x t r a c t t o a f l a s k i n which no e x t r a c t was added  at  first.  electric  C o l o r . . . i n t e n s i t y r e a d i n g s were made o n a K l e t t c o l o r i m e t e r u s i n g a green  w h i c h was 10  read a t a d e f i n i t e  time  A series  solutions  o f phenol  construction of a standard c o n t a i n e d 0.9  curve.  per ml.  were c a r r i e d  dilution.  and used  The p h e n o l  i n the  stock  solution 10  T h i s was d i l u t e d  m l . volumes o f the d i l u t e d  s o l u t i o n were a n a l y s e d f o r p h e n o l . dilution  The i n t e n s i t y i s  minutes a f t e r  was p r e p a r e d  mgms. o f p h e n o l  t i m e s , a n d 2 m l . t o 10  filter.  photo  stock  C o l o r d e v e l o p m e n t and  o u t as f o r a n a c t u a l p h o s p h a t a s e  deter-  mination. CATALASE - C a t a l a s e a c t i v i t y was e s t i m a t e d b y Sumner a n d Somer's m o d i f i c a t i o n o f t h e p r o c e d u r e 6,  and 9 m i n u t e i n t e r v a l s .  is  added to  (£2-).  2$ mis. o f i c e c o l d  o f Von E u l e r , u s i n g  Two m i s . o f t h e p l a n t e x t r a c t  catalase substrate at 0  time  when 5 m l . o f t h e m i x i s w i t h d r a w n q u i c k l y a n d b l o w n i n t o of and  2N s u l f u r i c  acid,  thus  9 minute i n t e r v a l s  enzyme-substrate  stopping  after  a drop  into  A t t h e 3,  6,  o f the f i r s t  o f hydrogen peroxide acid.  2N s u l f u r i c  acid.  The  i s arrested with the  Ten m l . o f a 5 p e r c e n t KI  solution  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 m i x , and then  o f 1 p e r c e n t ammonium m o l y b d a t e .  liberated  5 ml  a l i q u o t , o t h e r J> m l . a l i q u o t e o f t h e m i x a r e  addition of sulfuric cvs. added  the r e a c t i o n .  the withdrawal  w i t h d r a w n and b l o w n i m m e d i a t e l y decomposition  3»  iodine i s t i t r a t e d with  using starch solution The  0.00?N s o d i u m  3 minutes, the thiosulfate,  as an i n d i c a t o r .  v e l o c i t y constants  were c a l c u l a t e d u s i n g  After  the equation  K  v  f o rcatalase  activity  5 l / T l o g A/A-x.  The v e l o c i t y constant i s K, the minutes of digeXstion are T; A i s the t i t r a t i o n value of 5 ml. of the digest at 0 time i n terms of 0.005^ thiosulfate; A-x i s the t i t r a t i o n value after T minutes.  The various values obtained f o r K were p l o t t e d  against time and the graph was extrapolated to estimate the value of K at 0 time.  Catalase a c t i v i t y i s given i n the table  as the monomolecular value extrapolated to 0 time. Several modifications itf the method of Sumner and Somerfe.: were used i n this experiment.  The f i r s t modification  was that 2 ml. of extract and 25 ml. of substrate were used i n stead of the accepted 1 ml. of extract and 50 ml. of substrate. The second modification was made i n 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 i n 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 r e s u l t of experimentation using various concentrations of 3 percent hydrogen peroxide from 2.0 to 13.6 ml. i n 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 d i f f e r e n t substrates and the reaction mix with the highest a c t i v i t y used i n this experiment.  Catalase a c t i v i t y 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  concentration 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 o f the p r o c e d u r e o f s t a t t e r and co-workers. (£3).  will-  I t c o n s i s t s of incubating  plant  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 m e a s u r i n g i o d i m e t r i c a l l y the q u a n t i t y o f m a l t o s e formed.  Twenty f i v e m i s *  o f a 1 p e r c e n t s t a r c h s u b s t r a t e i s run f r o m a b u r e t t e i n t o a glass-stoppered added.  The pH  bottle.  Ten m l t . o f a 0.2  s h o u l d be 4*4-4.5*  M a c e t a t e b u f f e r Is  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 , w h i c h i s mixed and i n a 37°C. i n c u b a t o r f o r 20 minutes-.  A t the end o f  placed,  this  p e r i o d o f i n c u b a t i o n , 2 m l . o f N HOl\e*sa. blown i n and  the  s o l u t i o n mixed.  the  S i x t y m l . o f O.lN. WaDH faise added and  s o l u t i o n a g a i n r o t a t e d t o mix. p i p e t t e d i n t o the b o t t l e .  Twenty m l . o f 0.01  A f t e r 10 m i n u t e s , 10 m l . o f  p e r c e n t s u l f u r i c a c i d i s added and the mix i o n i s done w i t h 0.01  N IKI i s  rotated.  10  Titrat-  N sodium t h i o s u l f a t e . A b l a n k I s  by adding 1 m l . o f the enzyme a f t e r the a d d i t i o n o f the  run sul-  furic acid. The monomolecular r e a c t i o n c o n s t a n t ^ m y l a s e was  obtained K  s  from the  1/t  f o r beta-  equation:  l o g ' 0.1875 • 0.1875 - x  The  time i n m i n u t e s i s r e p r e s e n t e d  nected t i t r a t i o n  value.  by t , and x i s the  con-  &3.  INVERTASE - The method f o r t h e d e t e r m i n a t i o n o f i n v e r t a s e 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 b y J.B. Sumner and S.P. H o w e l l i n tho J o u r n a l o f B i o l , ohom, (&:4), was found to be i n a d e q u a t e  f o r t h e d e t e r m i n a t i o n o f i n v e r t a s e i n sugar  beet root e x t r a c t s .  T h i s method i n v o l v e s t h e use o f a s u c r o s e  b u f f e r e d s u b s t r a i n and t h e e s t i m a t i o n o f r e d u c i n g sugars p r e s e n t b e f o r e 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 s u c r o s e s u b s t r a t e i s n o t a p p a r e n t . The method used i n t h i s experiment s e p a r a t e methods. HLbbert,  i s a c o m b i n a t i o n o f two  One o f these was k i n d l y s u p p l i e d b y D.  o f t h e B r i t i s h sugar C o r p o r a t i o n L t d , i n P e t e r b o r -  ough, England. Oparin«s s t u d i e  The o t h e r was o b t a i n e d from a t r a n s l a t i o n o f s » }  The method i n use a t 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 C o r p o r a t i o n 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 r o o t s  are minced as r a p i d l y as p o s s i b l e and e q u a l w e i g h t s o f m i n c i n g a and w a t e r a r e b l e n d e d form f o r 5 m i n u t e s . cloth.  together w i t h excess c h l o r o -  The e x t r a c t i s f i l t e r e d t h r o u g h a f i n e  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 m l . o f the f i l t e r e d b e e t e x t r a c t ; 10 m l . o f a c e t a t e b u f f e r , pH 4.92 and an excess o f c h l o r o f o r m as»e added, and the tube shaken.  A f t e r the chloroform l a y e r  s e p a r a t e s , 10 m l . o f t h e s o l u t i o n ase p i p e t t e d i n t o a 50 m l . v o l u m e t r i c f l a s k , 10 drops o f b a s i c l e a d a c e t a t e (30°Be) added, and t h e m i x t u r e made up t o t h e 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 m i x t u r e i s f i l t e r e d and the p o l a r i m e t e r  r e a d i n g t a k e n i n a 200 mm.  tube.  The r e m a i n d e r o f the  b u f f e r e d e x t r a c t i n the s t o p p e r e d tube i s i n c u b a t e d f o r 48 hours a t 37° G. and the f i n a l p o l a r i z a t i o n i s d e t e r m i n e d  at  the end o f t h i s t i m e . If  the d e c r e a s e I n p o l a r i z a t i o n i n 48 h o u r s i s P,  then the enzyme a c t i v i t y (E.A.) i s e x p r e s s e d as: E.A.  =  5P W  I n v e r t a s e a c t i v i t y thus e x p r e s s e d i s assumed t o be 1000  times p o l a r i z a t i o n r e d u c t i o n b r o u g h t about by 1 m l . o f  e x t r a c t i n 1 h o u r a t 37°  C»  O p a r l n ' s method f o r the d e t e r m i n a t i o n o f 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 o f p u l p s.vse macerated  i n 200 m l . o f d i s t i l l e d w a t e r .  Two  a l i q u o t samples o f t h i s  s u s p e n s i o n are p i p e t t e d o u t ; one o f these i s i n a c t i v a t e d by adding c a l c i u m carbonate and b o i l i n g f o r f i v e m i n u t e s .  The  p a r a l l e l sample, n o t i n a c t i v a t e d , s e r v e s f o r the d e t e r m i n a t i o n of  invertase a c t i v i t y .  To a l l samples,  s u c r o s e i s added t o  the e x t e n t o f 20 p e r c e n t o f the w e i g h t o f p u l p t a k e n , t h i s b e i n g e s p e c i a l l y i m p o r t a n t , 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 u n t r e a t e d samples are m a i n t a i n e d a t 30° for  20 h o u r s , c l a r i f i e d w i t h l e a d a c e t a t e , and the r e d u c i n g  sugars d e t e r m i n e d .  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 invertase.  C«  The p r o c e d u r e 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 b e e t s a r e c l e a n e d and  t h e i r crowns s l i c e d o f f a p p r o x i m a t e l y \ i n c h b e l o w the d e f o l i a t e d beet surface.  The crown s l i c e s are minced i n a  g r i n d e r and 40 grams o f t h e s e - m i n c i n g s  aiss  b l e n d e d w i t h 100  m i s . o f d i s t i l l e d i c e w a t e r f o r 2 minutes..  The s u s p e n s i o n i s  f i l t e r e d t h r o u g h b r o a d c l o t h . One gram o f pure s u c r o s e I s p l a c e d i n each o f two, 250 m l , e r l e n m e y e r f l a s k s , and 25 m l , o f f i l t e r e d b e e t r o o t e x t r a c t d e l i v e r e d t o each f l a s k . s o l u t i o n s are mixed u n t i l t h e s u c r o s e i s d i s s o l v e d .  The  Ten m l .  o f a c e t a t e b u f f e r pH 4.92 aiiffi added t o the f l a s k s and t o one f l a s k a p p r o x i m a t e l y 1 gram o f c a l c i u m c a r b o n a t e .  The c a l c i u m  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 t a k e n t o a v o i d b u r n i n g t h e sugar.  Ten m l . o f c h l o r o f o r m tise  added t o 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 o v e r the s o l u t i o n s . a n d t h e y are i n c u b a t e d a t 37° C. f o r 48 h o u r s .  A t the end o f 48 h o u r s , t h e s o l u t i o n s a r e  a n a l y z e d f o r i n v e r t sugars u s i n g t h e somogyi/shaffer-Hartmann method (2-5). Enzyme a c t i v i t y i s e x p r e s s e d as mgms. i n v e r t sugars formed times 1000.  The b l a n k i n v e r t sugar v a l u e i s  s u b t r a c t e d from the a c t i v e r u n t o o b t a i n the number o f mgms. o f i n v e r t sugars formed b y 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 p u l p was weighed r a p i d l y on a b a l a n c e , p l a c e d I n an oven a t 110° C. f o r 10 m i n u t e s , and t r a n s f e r r e d t o 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 required three o r four  days and i t was n e c e s s a r y t o m i x t h e p u l p s e v e r a l t i m e s d u r i n g the d r y i n g p e r i o d .  Dry w e i g h t i s e x p r e s s e d on a p e r -  centage b a s i s . SUCROSE Sucrose was d e t e r m i n e d b y employing the method as o u t l i n e d i n t h e A s s o c i a t i o n o f A g r i c u l t u r a l c h e m i s t s (2/6). • I t i s the h o t w a t e r method o f d i g e s t i o n w h i c h p r o c e e d s as follows:  T w e n t y - s i x grams o f f r e s h o r f r o z e n p u l p (thawed  b e f o r e w e i g h i n g ) 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 w a t e r t o a 201.2 c c . K o h l r a u s c h flask.  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 p l a c e d i n a h o t w a t e r b a t h a t 80° c. at  The f l a s k i s r o t a t e d  i n t e r v a l s and s m a l l amounts o f 80° 0. d i s t i l l e d w a t e r a r e  added from time t o time so t h a t a t t h e end o f 30 m i n u t e s the water i n t h e f l a s k i s a l i t t l e above t h e mark. the  At t h i s time,  f l a s k i s removed from t h e w a t e r b a t h and the s o l u t i o n  a l l o w e d t o c o o l t o 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 v e r y 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 e t h e r .  E t h e r i s added t o reduce foaming,  i n t h e neck o f t h e K o h l r a u s c h f l a s k . p l e t e d t o t h e mark.  The volume i s com-  About 100 m l . o f t h e h o t w a t e r e x t r a c t  i s t r a n s f e r r e d t o a m e t a l cup o r a f l a s k c o n t a i n i n g o n e - h a l f gram o f l e a d a c e t a t e , shaken w e l l , and the s o l u t i o n a l l o w e d to  s t a n d u n d i s t u r b e d f o r a few m i n u t e s .  I t i s then f i l t e r e d  u s i n g 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  polar-  30. i z e d i n a 200 mm. tube.  A R e i c h e r t p o l a r l m e t e r was u s e d .  Readings a t room temperature a r e i n p e r c e n t s u c r o s e f r e s h weight,  s u c r o s e i s a l s o e x p r e s s e d on a p e r c e n t d r y w e i g h t  basis, INVERT SUGARS D e t e r m i n a t i o n s were made on 5 m l , p o r t i o n s o f t h e h o t water e x t r a c t used i n t h e a n a l y s i s f o r s u c r o s e , -The 5 m l , 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 K o h l r a u s c h f l a s k and d i d n o t c o n t a i n l e a d a c e t a t e . 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 p r o c e d u r e f o r t h i s method i s : P i p e t t e 5 m l , . o f sugar s o l u t i o n c o n t a i n i n g 0,2 t o 2,0 mgms, o f g l u c o s e 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 m l . o f i n v e r t sugar reagent.  M i x , s t o p p e r l o o s e l y w i t h a g l a s s s t o p p e r and p l a c e  i n a b o i l i n g w a t e r b a t h f o r 15 m i n u t e s .  Remove t h e t u b e ,  c o o l i n c o l d Water f o r 3 m i n u t e s u n t i l t h e temperature o f t h e s o l u t i o n i s 30-40° 0.  Add 1 m l . o f 5N s u l f u r i c a c i d , m i x ,  and a l l o w t o s t a n d f o r 2 m i n u t e s .  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 t h e I o d i n e h a s r e a c t e d . 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 t o c o l o r l e s s e n d - p o i n t . For  the b l a n k , use 5 m l . o f d i s t i l l e d w a t e r i n p l a c e 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 b y use o f the s&S c o n v e r s i o n t a b l e ,  i n v e r t sugars are  e x p r e s s e d i n terms o f b o t h p e r c e n t o f f r e s h and d r y w e i g h t s ^ ) o f pulp'.  32., IS.  ANALYTICAL METHODS ' DRIED AND GROUND PULP R  TOTAL NITROGEN  One  gram p o r t i o n s  o f t h e d r i e d and ground p u l p were  used t o d e t e r m i n e t o t a l n i t r o g e n b y t h e s t a n d a r d  Kjeldahl  method.  T h i s method, as d e s c r i b e d i n Loomis and S h u l l  requires  duplicate  powder.  The powder i s weighed on a 7 cm, f i l t e r p a p e r .  paper i s f o l d e d A selenlzed  1,000 gram samples o f t h e o r i g i n a l d r i e d  and d e p o s i t e d i n a 800 m l , K j e l d a h l  c r y s t a l I s added and 25 m l ,  s a l i c y l i c a c i d m i x t u r e poured i n . by The  (211)  frequently  flask,  of a sulfuric acid-  This a c i d mix i s prepared  adding 1 gram o f s a l i c y l i c a c i d t o 25 m l . flask i s rotated  The  of sulfuric acid.  f o r 30 m i n u t e s .  At t h i s time,  5 grams ~of sodium t h i o s u l f a t e a r e added and-the f l a s k warmed s l i g h t l y f o r 5 m i n u t e s , then c o o l e d .  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 t h e f l a s k and the m i x r o t a t e d .  Heat i s a p p l i e d g e n t l y  f r o t h i n g i s o v e r , and t h e n s t r o n g l y u n t i l c l e a r .  until A f t e r 30  m i n u t e s , t h e f l a s k I s removed from t h e b u r n e r and c o o l e d I n a fume c l o s e t , and  100 m l ,  250 m l .  o f w a t e r ase added, t h e f l a s k  o f 33 p e r c e n t NaOH p o u r e d I n ,  are n o t m i x e d ,  several  Inclined,  The a c i d and base  p i e c e s o f z i n c m e t a l a r e added.  The  f l a s k I s t h e n connected t o t h e d i s t i l l a t i o n a p p a r a t u s and rotated 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 m l , o f  0,14 N HOl s o l u t i o n .  M e t h y l r e d I s employed as an i n d i c a t o r .  A p p r o x i m a t e l y 150 m l .  o f d i s t i l l a t e sts& c o l l e c t e d .  used a c i d i s t i t r a t e d w i t h 0.14 N NaOH.  One m l .  The u n -  o f 0.14 N  33.  NaOH i s e q u i v a l e n t t o 1 mgm. o f n i t r o g e n .  Total protein  e q u a l s the number o f m i s . o f NaOH m u l t i p l i e d b y 6.25. gram o f s u c r o s e i s used i n p l a c e o f t h e powder f o r the T o t a l n i t r o g e n i s expressed is'.  One blank.  on a d r y w e i g h t b a s i s .  ANALYTICAL METHODS - ALCOHOL EXTRACT OP DRIED AND GROUND PULP SOXHLET EXTRACTION E x t r a c t i o n o f the d r y ground p u l p w i t h e l b a h o l 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  p l a c e d i n an e x t r a c t i o n t h i m b l e .  A loose cotton plug I s In-  s e r t e d and t h e t h i m b l e d e p o s i t e d 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 p e r c e n t e&footaol atsa added and an e x -  t r a c t i o n r u n f o r 45 m i n u t e s .  The e t h a n o l i s removed and 50 v  m l . o f f r e s h 70 p e r c e n t e t h a n o l a g a i n 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 h o u r s .  B e f o r e each ^ - t a ^ l  the e x t r a c t i o n cup i s a l l o w e d t o f i l l m a i n i n g i n the f l a s k I s r e p l a c e d .  change,  and the e&bohol r e -  A second 50 m l .  ethanol  p o r t i o n i s p u s e d a t the end o f the 2 h o u r 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 r u n o v e r n i g h t .  The c o m p l e t i o n o f s o l u b l e  sugar e x t r a c t i o n was d e t e r m i n e d b y w i t h d r a w i n g a h o l e x t r a c t surrounding  5 m l . o f efchs-  the t h i m b l e , a d d i n g 2 d r o p s o f a 15  p e r c e n t a l c o h o l i c s o l u t i o n o f a l p h a naptho1 and 2 m l . o f concentrated  sulfuric acid.  I n d i c a t e d incomplete  The p r e s e n c e o f a p u r p l e  carbohydrate  extraction.  ring  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 m l . t h i m b l e i s h e a t e d i n a 70° has  flask.  The  extraction  G. oven u n t i l the e x t r a c t e d  powder  d r i e d to c o n s t a n t w e i g h t . CLEARING AND The  DELEADING  a l c o h o l i c s o l u t i o n from the s o x h l e t  was  e v a p o r a t e d to 50 m l .  The  s o l u t i o n was  dissolved.  and  extraction  50 m l . o f d i s t i l l e d w a t e r added.  s t i r r e d u n t i l the r e s i d u e had b r o k e n up  T h i s h o t water e x t r a c t was  a t i v e l y t o a 250 m l .  volumetric f l a s k .  transferred  and  quantit-  A l l equipment  was  t h o r o u g h l y 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 w a t e r . The  e x t r a c t was  c o o l e d and  a c e t a t e s o l u t i o n added. was  -  2 ml. of a saturated,  A f t e r making t o volume, the  f i l t e r e d using suction.  as alum* o x a l a t e ated.  The  neutral  A p p r o x i m a t e l y 0.3  were added t o the f i l t r a t e and  d e l e a d e d s o l u t i o n was  pot-  the f l a s k r o t -  t e s t e d f o r an excess o f  dropped on the s u r f a c e o f the s o l u t i o n , and  i n t o a d r y f l a s k , the s o l u t i o n was  ox-  Toluene  the l i q u i d  to s t a n d 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 .  s u c r o s e and  extract  grams o f  a l a t e by adding 1 drop o f d i l u t e d l e a d a c e t a t e .  oantation  lead  was  allowed  After  de-  analyzed f o r  i n v e r t sugars.  INVERT SUGARS Duplicate  5 ml.  f o r i n v e r t sugar a n a l y s i s .  a l i q u o t s o f the e x t r a c t were u s e d The  d i l u t i o n f a c t o r i s 50.  Somogyi-Shafd!'er-Hartmann method d e s c r i b e d p r e v i o u s l y was p l o y e d . (25)  I n v e r t sugars are e x p r e s s e d on a p e r c e n t  The emdry  35,  w e i g h t b a s i s o f the o r i g i n a l d r y powder. SUCROSE F i v e m l . o f the d i l u t e d e x t r a c t u s e d f o r i n v e r t s u g a r a n a l y s i s was  p i p e t t e d i n t o a 100  t o volume w i t h d i s t i l l e d w a t e r . s u l f u r i c a c i d was The  e x t r a c t was  9.8  factor i s  One^half ml. of concentrated  by a u t o c l a v i n g  f o r 5 minutes at 5  T h e ' h y d r o l y z e d e x t r a c t was  N NaOH and 51ml• .  determination  f l a s k and made  added and 2 d r o p s o f m e t h y l r e d I n d i c a t o r .  hydrolyzed  pounds p r e s s u r e ;  ml. volumetric  neutralized with  dupMoate^aliquoTis".usedllniinvert,sugar  by the somogyi method.  The  total  dilution  1025. T o t a l sugars e q u a l s u c r o s e 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.  Total available  carbohydrates c o n s i s t s of t o t a l sugars p l u s The  a d d i t i o n o f t o t a l sugars and  starch-dextrins.  starch-dextrins i s  as b e i n g t o t a l a v i a l a b l e c a r b o h y d r a t e s . i s e x p r e s s e d as p e r c e n t o f rdegh w e i g h t ,  considered  T o t a l carbohydrate s u c r o s e and  Invert  sugar v a l u e s f o r a l c o h o l e x t r a c t e d p u l p are based on a  per-  c e n t o f d r y w e i g h t b a s i s o f the o r i g i n a l d r i e d powder. IT,  ANALYTICAL METHODS - ALCOHOL EXTRACTED PULP STARCH-DEXTRINS The  was  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  d e t e r m i n e d by the method o f Loomis and S h u l l .  residue (£7).  36. M a l t d i a s t a s e s o l u t i o n was Duplicate  employed.  1 gram p o r t i o n s o f r e s i d u e  i o n are t r a n s f e r r e d t o 250 m l . w a t e r use  added and  b o i l i n g water bath. to 35°  The method i s as f o l l o w s ; from the s o x h l e t  f l a s k s ; 50 ml o f  The  gelatinized starch solution i s percent malt diastase  added a l o n g w i t h s e v e r a l d r o p s o f t o l u e n e . G. o v e r n i g h t ,  ,Following i n c u b a t i o n ,  and  solution  flask Is held  the e x t r a c t s are f i l t e r e d i n t o 100 2 ml.  s o l u t i o n run i n t o each.  to volume and m i x e d . of potassium oxalate  The  cooled  rotated occasionally i f possible.  f l a s k s , the r e s i d u e washed, and l e a d acetate  distilled  the - f l a s k s h e a t e d f o r 30 m i n u t e s i n a  G. and 10 m l . o f a 0.5  a t Get 35°  extract-  of saturated The  ml.  neutral  s o l u t i o n s are made  D e l e a d i n g i s done by a d d i n g 0.3  grams  t o the s o l u t i o n s , w h i c h a r e a g a i n f i l t e r e d .  100 m l . o f the c l e a r e d and d e l e a d e d e x t r a c t s are h y d r o l y z e d  by  autoclaving  of  f o r 60 m i n u t e s a t 15 pounds p r e s s u r e w i t h 5 m l .  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  NaOH to m e t h y l r e d I n d i c a t o r , and Duplicate a t i o n by  5 ml.  with  the t o t a l volume d e t e r m i n e d .  a l i q u o t s are used f o r i n v e r t sugar d e t e r m i n -  the S&S method.  The b l a n k c o n s i s t s o f 50 m l . o f  0.1  p e r c e n t m a l t d i a s t a s e s o l u t i o n w h i c h i s h y d r o l y z e d as f o r s t a r c h and  the amount o f i n v e r t sugar p r e s e n t d e t e r m i n e d ,  starch i s  e x p r e s s e d as p e r c e n t o f d r y w e i g h t o f the erT^taotj une.x-tracked pulp using  the f a c t o r  0.90.  INSOLUBLE NITROGEN D u p p l i o a t e 1 gram p o r t i o n s o f the e x t r a c t e d were a c c u r a t e l y weighed and  residue  analyzed f o r i n s o l u b l e nitrogen  by  the s t a n d a r d 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 d r y 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 p l a c e d i n an 800 m l . K j e l d a h l  flask.  A s e l e n i z e d c r y s t a l i s added, and 25 m l . o f c o n c e n t r a t -  ed s u l f u r i c a c i d . fate mixture  E i g h t grams o f a sodium s u l f a t e - c o p p e r  anse s t i r r e d w i t h t h e a c i d and sample.  sul-  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 o v e r and t h e n s t r o n g l y u n t i l clear.  Heat i s c o n t i n u e d  f o r 30 m i n u t e s , when t h e f l a s k i s  removed from t h e b u r n e r r a c k and t r a n s f e r r e d t o t h e fume c l o s e t . A f t e r - c o o l i n g , 250 m l . o f w a t e r , 100 m l . o f 33 p e r c e n t and  NaOH,  s e v e r a l p i e c e s o f z i n c m e t a l are» p l a c e d 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 o u t 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 m l . o f 0.14 N NaOH i s e q u i v a l e n t t o 1 mgm. o f n i t r o g e n . One gram o f sucrose blank.  i s used i n p l a c e o f t h e powder f o r the  I n s o l u b l e n i t r o g e n i s e x p r e s s e d as p e r c e n t o f d r y  w e i g h t o f the ©]m<$fo-*i 4K&4c f **>&e-*rjlv.. v  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 t a k e n t o r e p r e s e n t  soluble nitrogen.  38.  i&.  RESULTS The f o l l o w i n g o b s e r v a t i o n s o f t h e e x t e r n a l  effects  o f f r e e z i n g and o f l e a f r e g r o w t h r a p i d i t y were n o t e d ; Immediately  f o l l o w i n g f r e e z i n g , the l e a v e s and p e t -  i o l e s became q u 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 green i n c o l o r . drooped.  The o l d e r l e a v e s were dead and d a r k  T h e i r p e t i o l e s were t h o r o u g h l y weakened and  The newer l e a v e s o f 1 i n c h h e i g h t i n t h e c e n t r e o f  the b e e t crown had w i t h s t o o d F o u r t h day -  freezing.  The f r o z e n l e a v e s were w i t h e r e d and no  r e g r o w t h observed on these b e e t s .  Regrowth from the crown  c e n t r e was b e g i n n i n g on the d e f o l i a t e d b e e t s .  r  E i g h t h day - No pathogens were p r e s e n t on t h e de-  crowned b e e t s .  R a p i d r e g r o w t h was o c c u r r i n g  on a l l o t h e r  treated beets.  Regrowth was removed f r o m the c o n t i n u a l l y de-  f o l i a t e d beets. E l e v e n t h day - New l e a v e s were c o n t i n u a l l y S i x t e e n t h day - The new l e a v e s h a d grown  appearing. strongly  ( 8 i n c h e s i n h e i g h t ) and r e g e n e r a t i o n o f t h e f o l i a g e was almost  complete. T w e n t i e t h day - Normal t o p appearance and complete  r e g r o w t h was observed.  The c o n t i n u a l l y d e f o l i a t e d  were s t i l l p r o d u c i n g abundant l e a f s h o o t s . beets  beets  The decrowned  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  their cut surfaces. Thus, i t appears t h a t the g r e a t e s t r e g r o w t h o f new l e a v e s o c c u r s from 4-17 days a f t e r f r e e z i n g , and t h e l a r g e s t sugar l o s s e s would be e x p e c t e d d u r i n g 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 e x p e r i m e n t were e x t r e m e l y f a v o u r a b l e f o r sugar l o s s and l e a f r e g r o w t h * 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 increase i n t h e i r s e v e r i t y a f t e r t h e i n i t i a l f o l i a r f r e e z i n g on t h e p r a i r i e s , thus p r o d u c i n g a s e t o f c o n d i t i o n s w h i c h were n o t p r e s e n t I n t h i s experiment.  New l e a f r e g r o w t h 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 t h e sugar l o s s e s o b t a i n e d would n o r m a l l y n o t 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 p e r c e n t d r y w e i g h t o c c u r r e d i n a l l treatments.  The l a r g e s t decrease was o b s e r v e d on t h e 1 6 t h  day a f t e r t r e a t m e n t .  The T / C v a l u e s f e l l I n the f r o z e n b e e t s  from 108.0 on t h e f i r s t day t o 80.0 on the 2 0 t h day; i n t h e d e f o l i a t e d b e e t s from 99.0 t o 79.0; i n the c o n t i n u a l l y def o l i a t e d b e e t s from 96.0 on t h e 4 t h day t o 78.0 on the 2 0 t h day; i n t h e decrowned b e e t s from 91.0 t o 77.0 on t h e 1 s t and 2 0 t h days r e s p e c t i v e l y . based on T / C v a l u e s a r e :  The;s© d e c r e a s e s as a p e r c e n t a g e F r o z e n 26.0 p e r c e n t ; d e f o l i a t e d 20.2  40. percent; continually d e f o l i a t e d 18.7 percent; decrowned 15.5 percent.  The highest l o s s i n percent dry weight occurred i n  the frozen beets and the l e a s t i n the decrowned beets,  percent-  age losses i n the d e f o l i a t e d beets are s i m i l a r to those f o r the frozen beets.  The control beets showed no decided reduction  i n dry weight during the 20 day period.  Dry weight r e s u l t s  are l i s t e d i n Table I I I . NITROGEN Total, soluble and insoluble nitrogen percentages are given i n Table IV. Total Nitrogen - The values i n a l l treatments decreased from the 1st to the 8th day.  The tendency a f t e r the  8th day was towards an increased amount of nitrogen measured as t o t a l nitrogen.  The T/C values r i s e above 100 f o r the  frozen, continually d e f o l i a t e d and decrowned beets.  Total  nitrogen i n 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 def o l i a t e d beets. the 8th day.  No general trends i n values were noted a f t e r  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  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  mm  18.4  91  20.2  . FRESH WEIGHT (26 GRAMS) BEET PULP EXPRESSED IN GRAMS DRY WEIGHT FROZEN  T/C  DEFOLIATED  T/C  CONT. DEFOLIATED  4  5.40  102  5.06  97  5.01  8  4.62  91  4.68  92  11  4.80  89  4.76  16  4.16  82  20  4.60  80  - CROWN  T/C  CONTROL  96  5.05  97  5.20  4.80  95  4.63  91  5.08  88  4.55  84  4.21  78  5.41  3.87  77  4.42  88  4.00  79  5.05  4.50  79  4.47  78  4.39  77  5.71  1 - T/C : PERCENT IN TREATED/PERCENT IN CONTROL X 100  T/C  8  I  i  -  t \l  1  V,  N  \  A' / / I  •  —  /  1 l_  A1  \\  \  7  V  it  A'  _\  \  K  \ \  i v / /  \  t  -/  /  \ w  \  V  //  /  \ \  //  \  \  /  \ \  \  i6  R.OZ  E:N  a  2o  DEF  i  /  \ \  -/ /  \  1 1-  U \ \ \  f-  •  i i-  /  /—  '\  •I-  1  \— -\  u. D  \  i  D.E.CRO  1 7,  / —y~  \  l  - | - ' — |  o.  v—  #  f-  _/  \  _l  T  // /  \  /  i  i  V±  Zo  "CD  ft  iA  /  i  1 | 1  /(b  /  /  /  1  IO 1  It D  1  c fONT  /-  1  1  -\ ist 1  1  |  .-<  3  r r  --  -  -  -a i  I  1  I"  EI  41. Soluble nitrogen - There was a decrease In soluble n i t r o gen i n a l l cases from the 1 s t to the 8th day.  A f t e r the 8 t h  day, the percentage of soluble nitrogen i n the treated beets was generally higher than i n the control beets. The d e f o l i a t e d beets were an exception to t h i s .  The T / C values f e l l and rose  as follows: values f o r the 1st, 8th, and 20th days - frozen 102.0, 45.0, 131.0; d e f o l i a t e d 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 o f sucrose based on fresh weight were: control 13.4; frozen 13.1; defoliated 12.3; cont i n u a l l y d e f o l i a t e d 12.5; decrowned 12.9.  The values obtained  when the percent sucrose f r e s h 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 r e s u l t s f o r the same days: d e f o l i a t e d 96.0 to 123.0; cont i n u a l l y d e f o l i a t e d 94.0 to 119.0; decrowned 111.0 to 121.0. Calculation of percent sucrose dry weight was done by determining the percent dry weight of the 26 gram pulp portions used f o r sucrose analysis and converting percent sucrose f r e s h weight to percent sucrose dry weight f o r each i n d i v i d u a l analysis. The sucrose values f o r 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 X TOT  XX  FROZEN XXX  INSOL  SOL  %  fo.  -  DEFOLIATED  -  TOT  T/C  INSOL  • - .  %  T/C  SOL  T/C  TOT  %  -  T/C  INSOL  • -• -  T/C • -  SOL  T/C  -  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  TOT.  T/C  1  INSOL.  T/C  DECROWNED  SOL.  *-  T/C  TOT.  T/C  INSOL.  T/C  SOL.  T/G  110  •  1  -  -  -  -  -  -  5.37  108  .394  93  4;98  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 l o s t the highest percent o f sucrose.  The continually de-  f o l i a t e d beets showed the next highest l o s s followed by the defoliated and frozen beets*  The percentages of sucrose  based on dry weight i n the alcohol extracts are lower than those obtained when peroent sucrose fresh weight i s expressed on a dry weight basis*  sucrose r e s u l t s are given i n Table V*  INVERT SUGARS Invert sugars, as analyzed f o r i n fresh pulp and expressed i n percent dry weight, increased i n amount following a l l treatments*  A noticeable r i s e i n i n v e r t sugars  occurred a f t e r the 8th day*  The T/C values on the 1st, 8th,  and 20th days were: frozen 87*0, 116*0, 140*0; d e f o l i a t e d 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  DAYS AFTER TREATMENT  CONTROL FROZEN fo FRESH WT. % T/C  Q_  13.20 13.90 13.90 13.20 13.90 13.20 13.20 12.50 12.90 12.50 fo  4 8 11 16 20  DRY WT.l 66.1 71.2 66.8 68.0 58.8  0  m  e  4 8 11 16 20  TREATMENT ~ DEFOLIATED CONTINUALLY DEFOLIATED fo T/C f T/c  fo  67.0 74.3 71.5 78.2 70.7  105.0 95.0 95.0 94.8 97.0 T/O  101 104 107 115 120  12.30 12.00 13.90 10.80 12.50 fo  63.4 66.7 76.0 72.5 72.2  m  •  m  93.2 86.5 100.0 81.8 97.0  12.00 12.00 12.50 13.90 12.00  91.0 86.5 90.0 105.0 93.0  fo  T/C  TJG  96 94 114 107 123  62.3 65.0 71.4 82.0 69.8  94 91 107 120 119  DECROWNED fo T/C #w •  «  14.20 13.20 13.20 12.00 12.00 %  ~  73.2 74.2 81.5 78.0 71.1  1- PERCENT SUCROSE FRESH WEIGHT CONVERTED TO PERCENT SUCROSE BASED ON THE DRY WEIGHT OF 26 GRAMS OF PULP.  108.0 95.0 95.0 91.0 93.0 T/C 111 104 122 114 121  TABLE V (CONT'D) SUCROSE CONTENT OF TREATED AND CONTROL SUGAR BEETS - ALCOHOL EXTRACT DAYS AFTER TREATMENT % PF.DRI^WT;CONTROL  % FROZEN T/C  DEFOLIATED T/C %  CONTINUALLY DEFOLIATED T/C %  1  79.0  75.5  96  67.0  85  -  u  73.3  77.9  106  76.4  104  60.7  8  66.0  65.2  99  76.3  116  11  68.9  61.8  89  64.4  16  67.4  61.7  92  20  71.6  58.8  82  DECROWNED T/C  %  67.8  84  83  68.5  94  61.7  93  64.8  98  94  59.0  .86  57.7  83  45.8  68  60.4  90  59.3  88  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  \\  /  \  \  \/fJ  1  ''I  /  //  / >  1  —  / / / 1//  * -  /-  lie  -/ —  /  ---  2  I  o )  T  t - r>  yL  i  —  —  '/  /  f  i  1I  1  i  / \ —  '  i  f  a< i  IT  1  1/  V  / \  1  I  /I'  \.  .11  i  A'  \  i  I -  /  k\  -i  1 1  A  za  g.  /  —  -  --  F R o z.E  s> 1  -  —t \  /  c\  I  / \  --•  -  -  i  Zo  1  <  t  c_ALLC 1 I  I  I!  | I  1  P  0. A T  |f  V  1  TV.  JUL&J I  -1 2( 0  •\  Ca  ..t= _v S.E a  1  RM  1 i  i  i  >  i  u l- T •=  -  f > f  "| ' 1  A r.l,0  d e c r o w n e d 117.0, 116.0, 2 3 2 . 0 . in  The p e r c e n t o f i n v e r t  the c o n t r o l b e e t s remained f a i r l y  rise  i n invert  s u g a r s was f o u n d  the d e f o l i a t e d b e e t s  The g r e a t e s t !  i n the decrowned b e e t s ,  showing t h e l e a s t r i s e  I n v e r t sugars, alcohol  constant.  sugars  as d e t e r m i n e d  with  i n inverts.  by a n a l y s i s  o f the  e x t r a c t o f t h e d r i e d b e e t p u l p , were i n c r e a s e d o v e r  the c o n t r o l s f o r t h e l a t t e r  16 d a y s i n t h e d e c r o w n e d and c o n -  t i n u a l l y defoliated beets.  The i n v e r t  sugar  content o f the  f r o z e n and d e f o l i a t e d b e e t s i n c r e a s e d t o t h e 4 t h d a y and t h e n a s l i g h t decrease  i s noted.  I n a l l a n a l y s e s , w h e t h e r made o n  the f r e s h p u l p o r d r i e d , p u l p , t h e a v e r a g e p e r c e n t a g e vert  sugars i n t h e t r e a t e d b e e t s i s g r e a t e r than  control beets, invert  of i n -  that o f the  sugar v a l u e s are g i v e n i n Table V I .  STARCH-DEXTRINS The  v a l u e s o b t a i n e d f o r the s t a r c h - d e x t r i n  o f t h e e x t r a c t e d b e e t p u l p do n o t i n d i c a t e c h a n g e s w h i c h have o c c u r r e d i n r e l a t i o n A general i n c r e a s e or decrease i s n o t noted  i n these r e s u l t s .  fraction  any q u a n t i t a t i v e  to the treatments.  i n the s t a r c h - d e x t r i n The p e r c e n t a g e  fraction  of starch i n  sugar b e e t r o o t i s n o 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 a r e given i n Table V I I .  TOTAL CARBOHYDRATES A decided decrease in  a l l treatments.  i n total  carbohydrates occurred  The l o w e s t d r o p was f o u n d i n t h e d e f o l -  TABLE VI  >  INVERT SUGAR CONTENT OF TREATED AND CONTROL SUGAR BEETS - AQUEOUS EXTRACT  DAYS AFTER TREATMENT  TREATMENT , CONTROL % FRESH WEIGHT 1  %  FROZEN T/C  -  1  m  DEFOLIATED T/C %  CONT. DEFOLIATED T/C %  DECROWNED T/C %  -  *•  -  -  m  IH  A  0.057  0.051  89  0.057  100  0.057  100  0.065  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  16  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  %  T/C  %  T/C  %  T/C  %  T/D  % OF DRY WT.  2  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  16  0.325  0.430  132  0.354  109  0.358  109  0.748  230  0.368 140 0.306 U6 0.264 1 - PERCENT OF FRESH WEIGHT OF ORIGINAL FRESH PULP. 2 - PERCENT DRY WEIGHT OF 26 GRAMS OF PULP.  0.690  262  0.610  232  20  TABLE VI (CONT'D) INVERT SUGAR CONTENT OF TREATED AND CONTROL SUGAR BEETS - ALCOHOL EXTRACT  DAYS AFTER TREATMENT  CONTROL % DRY WEIGHT  1  %  FROZEN T/C  TREATMENT DEFOLIATED % T/C  CONT. DEFOLIATED T/C %  DECROWNED % T/C  2.0  95  -  -  1.9  91  172  2.2  122  3.1  172  2.3  128  4.1  HI  1.1  38  3.1  107  4.2  H5  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  2.1  1.8  A  1.8  3.1  2.9 11  1 - PERCENT DRY WEIGHT OF ORIGINAL DRIED PULP  — /  -  t  A,  si  I  1  /  \  i i  / /  /  -'  t  /  I  1  II V, 1  t  >  •1  \  \  //  A.  \  \,  //  \ \  7 \  •  /  —  \  I  b  \  . 0  *  L  ~  4  .\  i  I  iI  \  \  \  t  J  /  \  \\  -  /  i  -  »-  A  •i  f y  / /  X  f  r  / /  /  i\ T/  1,  1  j  /  <  •  i  1  1  o  o -<  V  \ /  •o  >_( Zt  _  >  -1A ~y  V  \ \  r  ,0  J  8  /  \  7(\  F  /  7 I  1  1 It,  /  '•  \ \  ~~\  1  1  /•  —  _  \  —/  \<-  \  J  \  Zc  l<o  \  >  •A  H9  I  —  -'  0  f  \  | 1 1 D i e ..f  1 ML  Co  1  Eh  -r c  A L C O  f  Jl  J  H  l_l __ OL  -<  ) T..  EXTRACT])  r_ _  Lf  Xn  •J X  -  H  1A  -  =--1 o  e ^ r-t -G —  0-  TABLE VII STARCH - DEXTRIN CONTENT OF TREATED AND CONTROL BEETS  DAYS AFTER TREATMENT  CONTROL % DRY WEIGHT  1  %  FROZEN T/C  DEFOLIATED % T/C  CONT. DEFOLIATED DECROWNED % T/C % T/C  1  0,528  0.528  100  0.383  73  -  -  0.440  84  4  0.309  0.445  144  0.258  84  0.437  142  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  16  0.485  0.420  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.  i a t e d beets, while the decrowned beets have the highest drop. Total carbohydrate values are given i n Table VIII. qre  based  on  alcohol  fne values  extraction-  ENZYMES PHOSPHORYLASE In the second experiment of October. 1954, phosphorylase a c t i v i t y appeared to decrease with time i n a l l treatments.  A c t i v i t y i n the control beets, e s p e c i a l l y from the 11th  day was considerably higher than i n the treated beets.  There  was a sudden decrease i n phosphorylase a c t i v i t y from the 1 s t to the 4th day i n the treated beets.  The results of experiment  II are given i n Table x» PHOSPHATASE Phosphatase  a c t i v i t y decreased from the 1st to the  11th days i n a l l treatments except decrowned.  An increase i n  - a c t i v i t y occurred between the 11th and 20th days.  In the  decrowned beets, phosphatase a c t i v i t y Increased s l i g h t l y towards 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 f o r each group were: frozen 550, 340, 460; def o l i a t e d 520, 350, 440; continually d e f o l i a t e d 480(4th day), 350, 440; decrowned 530, 380, 410; control 450, 370, 450. Although the a c t i v i t y o f phosphatase f e l l i n the control beets, the decrease i s not as-high as that i n the treated beets. The r e s u l t s are given i n Table IX.  TABLE VIII TOTAL CARBOHYDRATE ( SUCROSE, STARCH-DEXTRINS, INVERT SUGARS) CONTENT OF TREATED AND CONTROL SUGAR BEETS  DAYS AFTER  LTMENT  TREATMENT  CONTHOL % DRY WEIGHT  1  FROZEN % T/C  DEFOLIATED % T/C  CONT. DEFOLIATED T/C %  81.6  77.8  95  69.4  85  mm  4  75.4  81.4  108  78.9  104  64.3  8  69.5  69.7  100  77.7  112  11  71.9  65.6  92  69.0  16  70.7  65.4  93  20  75.2  62.9  84  1  '  -  DECROWNED % T/C 69.1  85  86  71.2  95  65.0  94  69.2  96  96  63.1  88  62.2  87  49.9  71  63.6  90  63.8  90  65.1  87  66.6  89  59.8  79  A U O O H O L .  EXTRACTION  1 - PERCENT DRY WEIGHT OF ORIGINAL DRIED PULP - BASED  1b  u  -M  -/  \vr h \ \  //  V  /  \\  /  "\ \\  f  _  4-  1  \  / • \ Is  l_  //-I  \  \U  1  1L  \-  / I1 V'  f  u  /-  ^—  /  \  /  \^ \  I  1 lb  -  -  1 1  D I.L(  ..i  )  r_  8.1  ©  1 / •  I I  \\  // /'/  /  //  V  f  \  /  \  _l /J  h  /  1  -/  i  1  1  V 1  \  /  L\, — /  I  \-  ZO  1/  i  i  I I I ! .1  co A L CAK O O HY  Ta  1  to  lla  E.•  I  ~/  i  1  '/  \  N  —  N  \  \\  /-  D.E O-  i  i  \  • V  '-  \L\ L. Nr i i  1  -- —  •-  V  ./.  -  zo  ib  a. Z . . E . N  /  /  i  2o La.  " / /  N  r  bF  Q  1  —1— A T _E  D  ——-  7,  t  ' T•a  T —\— FZHT 1  •  Cit  e M E D.  TABLE IX ROOT ENZYME A C T I V I T I E S OF TREATED AND  CONTROL SUGAR  BEETS - EXPERIMENT I  TREATMENT ENZYME  DAYS AFTER TREATMENT  FROZEN_T/C ACT. 1  PHOSPHATASE  B " AMYLASE  1 - ENZYME  1  DEFOLIATED T/C AAGT.  CONT.DEFOL T/G ACT.  1  550  125  520  116  4 8  500 430  112 98  480 450 350  107 102 95  480 430 350  107 98 95  ±  3  4  0  9  2  -  "  - CROWN T/C ACT. 530  CONTROL AGT.  118  450  540 120 400 91 380 103  450 440 370  16  400  100  410  103  430  108  360  90  400  20  460  102  440  98  440  98  410  91  450  5To3  xTo3  1  0.49 197  0.40  160  4  0.16  41  0.18  46  8  0.32 140  0.27  H  0.28  61  16  0.30  20  0.18  ACTIVITY  5To3 -  xTo~3  xlo~3  -  0.60  240  0.25  0.23  59  0.34  87  0.39  118  0.37  160  0.21  91  0.23  0.28  61  0.23  50  0.33  72  0.46  40  0.33  44  0.35  44  0.49  65  0.75  60  0.28  93  0.42  140  0.33  110  0.30  TABLE IX (CONT'D) ROOT ENZYME A C T I V I T I E S OF TREATED AND CONTROL SUGAR BEETS - EXPERIMENT I  TREATMENT ENZYME  DAYS AFTER TREATMENT FROZEN T/C ACT. 1  xlO  CATALASE  1  DEFOLIATED T/C ACT. xlO  2  CONT.DEFOL T/C - CROWN T/C CONTROL ACT. ACT. ACT. xlO  2  xlO  2  xlO  2  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  11  0.9 7  79  0.74  60  1.08  88  1.0 7  87  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  - ENZYME A C T I V I T Y  1,  D  o  1  \  /  // ' /'/ '/  K  Hi  \  \  u  \  —\  -\-  V  /  1  —>  //  h  /•/  l_  t—  -1—  v. ^\  1 \ A  -t  \\ \\  h •v _i  1r  r _4-  //  V"  \\ /  /  />  J \\  y  /  A '  -  1 \<o  f/  'P  >.z._e.r>  O 1 /•  —  t 1  ZO  D t F o.  1 T  1  '/  _E .0  ft  \  i  \  _  t  4  / />  \  // /y  /  \\  zO Ff  1  /I /  \  /  \  r V  //  _  A \  \  t  /[  1  f  /l  \j  1  ©  j _  v"  ,'  /  /  \  \ r 1 lb  'I / 1  //  " x~ \i —  Ik j D.E..CR.C  /. Sl.t.O  n X  D  X  e  t  A C J : l_ \/_ i"r:  5.1.1  20 1  1 1  - c  r\  - -  /c -  i  -r-  7( - C O  •  E-  >-  t  B  /  b  \  1  i i  i  \  —  N  /  i J }/  \  \  \  I_  i  \ \  A-i.  ^—  1  1  V  A •  \ \  /i  N  / y—  \  -  11"  •  o  i  lb  z£  0.e  5  i —)i—  p  i  \  1 /LLJ  i  V  r  _C  D  ft  AI —  i  i  7"  i  \  \  V  r  /1  \  i  /  11  —  - V  to  -_  — -V  1  lb  \  •  \  1  f  A  1  i 1  ~t  \ \  1  i  •  1  -A  A/  -V  1  II  /  -\  \  c  Z-<  jIS //  \ \  Sr  •-  -  1  Die  ..R.C  \  ---  —  -•  1  |  1 20  lb  A  1  Cc * -T  >a£..d>  Q1  1!  :t  E -A T  - V T T1  \z  De  £  L.  | |  1  vi  I  _! <- X  •-  .1. 5<  o  D  4-5 BBTA-AMYLASB No changes occurred f o r beta-amylase a c t i v i t y which could be d e f i n i t e l y attributed to starch degradation. The values found i n both experiments (August and October) are not similar.  There was a tendency f o r beta-amylase a c t i v i t y i n  the treated beets to be lower than that o f the controls i n experiment I but this relationship was not duplicated i n the 2nd experiment*  The r e s u l t s o f experiment I are given i n Table IX,  and those o f experiment I I i n Table X. CATALASB Catalase a c t i v i t y increased i n the frozen beets from the 1st day to the 11th day and then decreased towards the 16th day.  The pattern i n the d e f o l i a t e d beets was similar to  that f o r the frozen beets - that Is, an Increase i n a c t i v i t y to the 11th day following treatment and then a general decrease.  In the cohtinously d e f o l i a t e d beets, the catalase  a c t i v i t y on the 4th and 8th days was higher than i n the contr o l beets.  There was also a decrease i n a c t i v i t y from the 11th  to the 20th day i n t h i s group*  The a c t i v i t y decreases as  would be expected, i n the decrowned beets.  The results are  given i n Table IX* iNYERTASB $he results o f experiment I I are given i n Table X* Invertase a c t i v i t y increased i n the frozen beets and decrowned beets from the 1st to the 8th day a f t e r treatment.  Activity  46  of the enzyme on the 15th day was greater In a l l treated beets than i n the controls*  Invertase a c t i v i t y could not be  detected i n the continually d e f o l i a t e d beets on the 1st and 12th days following treatment.  A c t i v i t y 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 -  ENZYME  INVERTASE  PHOSPHORYLASE  DAYS AFTER TREATMENT  EXPERIMENT I I  TREATMENT FROZEN ACT.  T/C  DEFOLIATED ACT.  1  130  100  90  70  A  610  185  830  252  8  810  262  290  12  170  85  15  240  1  CONT. DEEOL ACT.  T/G  - CROWN ACT.  T/C  CONTROL ACT.  0  0  170  131  130  390  118  370  112  330  93  190  61  420  136  310  140  70  0  0  110  55  200  218  360  326  240  218  560  520  110  56  400  72  515  69  490  33  236  14  4  34  74  17.5  38  21  A6  13  28  46  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  T/G  TABLE X (CONT'D) ROOT ENZYME ACTIVITIES OF TREATED AND CONTROL SUGAR BEETS - EXPERIMENT II  ENZYME  DAYS AFTER LTMENT  TREATMENT FROZEN T/fc DEFOLIATED ACT. ACT.  T/C  1  xlO^  BETA AMYLASE  xL03  CONT. DEFOL. ACT.  T/C  xlO^  T/C  xl03  1  0.28  65  0.29  67  0.39  91  4  0.32  266  0.30  250  0.27  225  8  0.23  92  0.16  6A  0.012  12  o.u  52  0.23  85  15  0.28  108  0.29  112  1 - ENZYME ACTIVITY  - CROWN ACT..  0.34  CONTROL ACT.  xlO? 79  0.43  -  0.32  266  0.012  0.12  48  0.25  0.25  92  0.27  100  0.27  0.33  127  0.34  131  0.26  47  INVERTASE ACTIVITY IN VARIOUS REGIONS OP THE BEET ROOT The highest a c t i v i t y value found was In the crown portion o f the beet root.  Invertase a c t i v i t i e s i n other  regions i n order of decreasing a c t i v i t y were: beet centre 290; outer surface 279; beet t i p 210. The crown had an act* i v i t y o f 300 E.A. u n i t s .  The values r e f e r to milligrams o f  Invert sugar difference between the actual run and the blank m u l t i p l i e d by 1000.  The r e s u l t s are l i s t e d i n Table I .  VALIDITY OF THE SAMPLING METHOD These r e s u l t s showed that the 40-gram aliquot o f beet pulp which was drawn from the t o t a l weight o f pulp cons t i t u t e d a representative sample.  The values obtained f o r  each aliquot agree c l o s e l y with one another.  The  t  tt  H  value  for these r e s u l t s i s small, i n d i c a t i n g that the sampling error i s n e g l i g i b l e .  The results are given i n Table I I .  TOP WEIGHTS AND ROOT VDEIGHTS AS RELATED TO SUGAR CONTENT No c o r r e l a t i o n could be found when the average top weights of each group of d e f o l i a t e d beets were compared to the average root weights and sugar content o f those groups. Also, no c o r r e l a t i o n could be found between percent sugar and average root weight, Table XI*  some t y p i c a l r e s u l t s are given i n  TABLE XI  COMPARISON OF AVERAGE LEAF WEIGHTS TO AVERAGE ROOT WEIGHTS AND SUGAR CONTENT AT .HARVEST TIME (RESULTS GIVEN FOR DEFOLIATED GROUP) pay a f t e r treatment  Average l e a f wt.  Average root wt.  1  415 gms  180 gms  nil  . -  4  422  n  474  II  nil  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  1 - AVERAGE OF 10 BEETS*  Average wt. o f regrowth  % sucrose f r e s h wt*  48.  9,  DISCUSSION Tne results of this experiment indicate that there i s  no r e l a t i o n between average l e a f weight and average sucrose content.  Also, there i s no r e l a t i o n between average root  weight and average sugar content i n mature beets* Both l e a f and crown removal cause a pronounced decline i n the y i e l d of dry matter of the root*  Tne peroent  dry weight decreases with time following treatment*  percent  dry weight decreases i n the continually d e f o l i a t e d and def o l i a t e d beets are similar, and indicate that the newly formed attached leaves do not constitute the only source of dry weight l o s s *  The d e f o l i a t e d beets exhibited a l a r g e r dry  weight decrease than the continually d e f o l i a t e d 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 l o s s of the other treated groups* The following conclusions are made with respect to dry weight decreases i n beet root tissue a f t e r f o l i a r l o s s : 1*  Frost may i n i t i a t e a higher rate of meristernatic  a c t i v i t y i n beets, thus increasing l e a f regrowth, sucrose loss and dry weight decrease* 2*,  A decrease |n dry weight i s either related to  a loss of t o t a l s o l i d s , as sucrose, which i s used i n l e a f r e growth, or an increase i n the hydration o f the beet root  49. tissue.  Dry weight l o s s i s l i k e l y a r e s u l t of both a sucrose  disappearance and water increase i n the beet root, these substances being u t i l i z e d i n l e a f 3.  regrowth.  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  t o t a l carbohydrates i s lower i n the decrowned beets.  I t may  be that the low decrease i n dry weight i s an e f f e c t caused by the evaporation of moisture from the cut root surface. The average percent fresh weight of sucrose i s lower i n each treated group than i n the control,  A 0.1 percent  sucrose difference can be detected with the polarimeter used i n the experiment.  The.duplicate analyses consistently checked  within 0.1 percent. I t i s observed that the T/C values i n a l l treatments r i s e above 100 a f t e r the 8th day when percent sucrose f r e s h weight i s converted to dry weight percent,  percent sucrose,  expressed by t h i s method, increases as the percent dry weight decreases.  The decreases i n percent sucrose f o r each period  after treatment do not equal the corresponding dry weight l o s s . 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 f o r the alcohol extract are as follows; frozen 94.0; d e f o l i a t e d 92.0; f o l i a t e d 88.0; decrowned 87.0.  continually de-  These r e s u l t s indicate that a  50. sucrose l o s s has occurred i n the treated beets. F o l i a r freezing, continual or a single d e f o l i a t i o n , 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 a f t e r freezing and defoliation,  since the most rapid l e a f regrowth was  apparent  during t h i s period i n these beets, 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. These r e s u l t s correspond with those of other workers (5) who have noted the r e l a t i o n between l e a f regrowth and percent sugar l o s s . Leaf regeneration was complete at the end of 20 days following f o l i a r l o s s and i t would be expected that the percent of sucrose i n the beet root should r i s e a f t e r t h i s 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. I t was noted that new l e a f shoots were a c t i v e l y appearing on the continually d e f o l i a t e d beets a t the 20 day point.  This leads to the conclusion that the region of high-  est sucrose disappearance i s i n the beet crown, l i k e l y through increased r e s p i r a t i o n , and that whether or not the new leaves are removed, the meristematie a c t i v i t y i n the crown continues at a higher l e v e l than In the normal growing beet.  Removing the meristematlc centre or crown from a beet  5 1  •  would, I t was thought, reduce sucrose l o s s considerably, i t was found that sucrose loss i n the decrowned beets i s higher than i n 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 a c t i v i t y  i n the decrowned beets was higher than i n the control beets, p a r t i c u l a r l y f o r the f i r s t 8 days a f t e r treatment. An increase i n the amount of i n v e r t sugars present occurred following freezing, d e f o l i a t i o n or decrowning.  Al-  though an inversion of sucrose would be accompanied by a r i s e i n invert sugars, i t may be that the inverts formed are i n t e r mediate and transitory i n metabolism.  The percent increase i n  invert sugars does not equal percent sucrose l o s s , suggesting that the glucose and fructose 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 withi n certain parts of the beet root - most probably the crown. I t 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 i n v e r t 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  s l i g h t inversion of sucrose during alcohol extraction. Since the t o t a l amount of reducing sugars i s f a i r l y constant throughout the growth of the beet (11), any increase i n invert sugars should be attributable to some d e f i n i t e b i o -  52.  chemical change or changes.  I t i s possible, and supported by  other workers (25), that sucrose i s inverted to the reducing sugars which move from the root c e l l s 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 v i s i b l e l e a f r e growth has occurred (1),  This i s substantiated by the sugar  loss i n the decrowned beets.  In a l l other treatments, abundant  l e a f shoots were seen appearing a t 3 days time following treatment.  The conclusion i s that sucrose l o s s i s not  necessarily accompanied by v i s i b l e l e a f regrowth.  The con-  clusion reached from the r e s u l t s o f t h i s experiment i s that an increase i n i n v e r t sugars does occur following f o l i a r l o s s , and that i t i s a t the expense of the stored sucrose. The percent o f starch i n sugar beet root i s low, as the beet synthesizes sucrose as i t s main storage product.  The  results f o r "starch-dextrins do not indicate polysaccharide synthesis at the expense of sucrose but the T/C values suggest a l o s s o f starch.  The beta-amylase  and starch phosphorylase  a c t i v i t y i n beet root tissue i s low, which might indicate that the starch synthesizing and degradation systems i n sugar beet root are not as e f f i c i e n t as they are i n other plant tissues, as f o r 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 r e l a t i v e l y high i n the root c e l l s .  The conclusion i n t h i s  53. experiment regarding starch-dextrins i s that they are of no d i r e c t importance to sucrose l o s s a f t e r f o l i a r removal e i t h e r by f r o s t or manual d e f o l i a t i o n .  The average percent of dry  weight starch-dextrin values i n the controls i s higher than the corresponding average f o r the treated beets, hence a decrease i n the amount of staroh i n the treated beets i s suggested* The decrease i n percent of t o t a l carbohydrates follows the f a c t that sucrose disappears,  since sucrose l o s s  and invert sugar increase occurs, then not considering starchdextrin synthesis or breakdown, the percent of t o t a l carbohydrates would at f i r s t be expected to remain constant*  A  measurement of t o t a l carbohydrates i n beet root tissue when l e a f regeneration i s occurring would l i k e l y give a value lower than that f o r a normal beet since part of the i n v e r t sugar present as a r e s u l t of sucrose inversion i s being used i n catabolism.  This would lower the amount of carbohydrate  i n the beet root tissues, hence t o t a l carbohydrate estimations provide a reasonable basis f o r determining both the amount of sucrose l o s s and the time a f t e r treatment when the highest l o s s occurs.  I t i s not known from the r e s u l t s of t h i s work  i n which region of the beet root the highest t o t a l carbohydrate reduction i s found* An increase i n soluble carbohydrates would favour an increase i n nitrogen i n the beet root*  The p r i n c i p l e  54. regions of protein synthesis In plants are i n meristems or i n storage tissue, p a r t i c u l a r l y the l e a f .  Amino acid synthesis  may  These amino acids  occur i n the roots of some plants.  may  be translocated from the tissues i n which they originate to distant tissues before being converted i n t o proteins, meristematic centres, protoplasmic from amino acids.  proteins are  in  constructed  Amino acid synthesis can not occur without  an adequate supply of carbon compounds,  primary synthesis  of amino acids and s i m i l a r compounds occurs only at the  ex-  pense of carbohydrates or t h e i r derivatives, which serve together with nitrogen containing compounds as a source of energy.  Rapid amino acid synthesis r e s u l t s i n a diminuition  i n the amount of carbohydrates i n a plant  (30).  Growth of meristems requires carbohydrates and n i t r o gen.  Both of these kinds of compounds are assimilated i n  r e l a t i v e l y large quantities, e s p e c i a l l y during the c e l l d i v i s i o n and enlargement phases of growth.  Considerable  amounts of carbohydrates are also used i n the process of r e s p i r a t i o n i n any a c t i v e l y 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 l o s s and active l e a f regrowth; 1.  since l e a f regrowth requires protein synthesis  which i n turn requires: amino acid synthesis, then carbohydrate i n the growing plant w i l l be r a p i d l y 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 i n h i b i t organic nitrogen synthesis* 3.  An increase i n organic nitrogen could be  ex-  pected i n the crown of the beet which i s r e s p i r i n g more than the other portions of the root*  An increase In i n v e r t sugars  occurs i n beets which are i n the stage of active l e a f regrowth*  An i n v e r t sugar increase favours amino acid synthesis* 4.  A decrease i n organic nitrogen could be expected  i n those regions where carbohydrates are disappearing r a p i d l y * 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 nitrogen.  analyzed f o r  The nitrogen values are not, then, those of the  main active growing or l e a f producing region of the beet. Total nitrogen decreased from the 1st to the 8th i n a l l treated beets*  The decrease i n nitrogen may  day  be  associated with a translocation of amino acids out of the region of the beet root analyzed*  A measureable r i s e i n  t o t a l nitrogen occurred a f t e r the 8th day and i t was a f t e r 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 translocated to the active growing regions.  Inorganic nitrogen i n  the form of n i t r a t e 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 d e f o l i a t e d . crease In soluble nitrogen may  This i n -  possibly be associated with  the observed increase i n invert sugars,  such soluble n i t r o -  genous substances commonly found i n the beet root as glutamine, asparagine, betain and a l l a n t o i n may  be  synthesized.  Insoluble nitrogen decreased In amount from the 1st to the 8th day In the _ •  d e f o l i a t e d and decrowned beets.  Insoluble nitrogen consists of organic nitrogen as proteins. A decrease i n insoluble nitrogen would mean an increase i n soluble protein and other nitrogenous compounds as amino acids f o r 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 t h i s experiment, 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 a c t i v i t y decrease suggests that the - synthesis o f starch i s reduced,  since 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 o f the tissue, a decrease i n a c t i v i t y would possibly be i n s i g n i f i c a n t ,  increased r e s p i r a t i o n due  to l e a f regrowth would favour the use o f glucose-phosphate as a respiratory substrate i n new tissue formation and not starch synthesis* phosphatase a c t i v i t y decreased i n a l l treated beets from the 1 s t day to the 11th day and Increased between the 11th and 20th days.  The action o f plant phosphatase i s the  s p l i t t i n g o f f o f 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 i r r e v e r s i b l e enzymatic reaction. A decrease i n phosphatase a c t i v i t y would r e s u l t i n l e s s substrate being s p l i t into inorganic phosphate and glucose.  The e f f e c t o f this i s that more phosphorylated  glucose Is available f o r r e s p i r a t i o n .  The reason f o r a  lower apparent phosphatase a c t i v i t y i n treated beets i n t h i s  58. experiment l a not known.  I t may  be associated with an i n -  creased respiratory rate. Although beta-amylase a c t i v i t y i n sugar beet root was  detected,  the a c t i v i t y was  very low, requiring incubation  of the enzyme with the substrate f o r a longer period of time than i s normally used i n the estimation.  No  correl-  ation can be found between starch-dextrin content and amylase a c t i v i t y i n t h i s experiment,  since the starch-dextrin  f r a c t i o n of beet root tissue Is low,  then beta-amylase  might be of some importance i n maintaining  a low starch  content i n root c e l l s thus making glucose available f o r sucrose synthesis.  I f , as reported  (13), starch i s syn-  thesized i n beet root c e l l s only when the sucrose eon* 1 centration becomes high, then a high concentration  of  sucrose might be associated with a low beta-amylase a c t i v i t y . The reverse of this could also be  acceptable.  Tyson (8) found that catalase a c t i v i t y i n beet leaves i s p o s i t i v e l y correlated with vigor and growth i n sugar beet plants.  Metabolically active plant tissues  usually have a high rate o f r e s p i r a t i o n and a correlated high enzymatic a c t i v i t y .  Measurements of the catalase  a c t i v i t y of a tissue are therefore often used as an index of the i n t e n s i t y of metabolic a c t i v i t y i n that t i s s u e . The apparent a c t i v i t y of catalase should  increase  i f a higher l e v e l of r e s p i r a t i o n r e s u l t s i n the beet root as  59.  a consequence o f f o l i a r l o s s ,  since r e s p i r a t i o n rates i n  sugar beets Is r e l a t i v e l y higher In the growing crown than i n the older region o f the beet, 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  respiration rate i n the maturing tissues decreases with age, the catalase " a c t i v i t y index" becomes lower.  A higher  catalase a c t i v i t y might be expected i n the beet crown following f o l i a r l o s s and active l e a f regrowth. f o r catalase determination  The tissue used  was sampled from the diagonal  sections of the beets and the crown portion o f the root was not included. An increase i n a c t i v i t y was found f o r a l l treatments when the T/C values are used as the basis o f a c t i v i t y determination*  The increase occurred from the 1 s t to the 8th day.  When the actual monomolecular values f o r each day a f t e r t r e a t ment are compared, i t i s seen that catalase a c t i v i t y i n each group decreases as follows: from the 1 s t to the 16th day i n the frozen beets; from the 1st to the 11th day i n the def o l i a t e d and continually d e f o l i a t e d beets; from the 1 s t to the 8th day i n the decrowned and control beets* The sudden increase i n catalase a c t i v i t y , based on T/C values might indicate a higher l e v e l of r e s p i r a t i o n i n the mature beet root c e l l s , the energy o f r e s p i r a t i o n used i n the movement o f soluble substances through the c e l l membrane* Based on actual monomolecular values, catalase a c t i v i t y  60. decreases with time i n the older c e l l s of the beet root following f o l i a r l o s s * prom the results of the second experiment, a few correlations can be made between invertase a c t i v i t y and sucrose content,  invertase a c t i v i t y Increased over the con-  t r o l s i n both the f o l i a r frozen and decrowned beets following treatment.  The highest sucrose l o s s based on dry weight  occurred i n the decrowned beets, suggesting a c o r r e l a t i o n , as proposed by oparin (7), between sucrose content and i n v e r t ase a c t i v i t y *  A p o s i t i v e c o r r e l a t i o n between invertase  action and sucrose content i s not evident In both groups of defoliated beets. I t i s well known now that the enzyme invertase i s actually present i n beet root tissue, but i t s r o l e i n sucrose metabolism i s unknown.  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 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 f i e l d i n May, 1954*  The young beets were transplanted to  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 o f plants* treatments and a control were used* oft  pour d i f f e r e n t  The treatments consisted  f o l i a r freezing; a single d e f o l i a t i o n ; continual de-  f o l i a t i o n ; decrowning*  s i x groups o f beets, or a t o t a l o f 60  beets, received one type o f treatment.  This was repeated f o r  each treatment giving a t o t a l of 300 beets used Including the controls. Freezing was accomplished using dry (C02) i c e , and d e f o l i a t i o n by knife removal of the leaves approximately £ inch above the beet crown.  The decrowned roots were s l i c e d  immediately below the p o s i t i o n 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 a f t e r 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 s l i c e d o f f . 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 w e l l mixed*  The removed leaves were weighed along with the roots  harvested* Analyses were c a r r i e d out on fresh beet pulp f o r catalase. phosphatase, phosphorylase,, invertase, beta-amylase, sucrose, Invert sugars and dry weight,  some o f the fresh  beet pulp was stored i n a -10°c r e f r i g e r a t o r f o r future analyses. In the second experiment,  the diagonal section o f  the beet root was used f o r beta-amylase and phosphorylase determination, while a crown s l i c e was employed i n the estimation o f invertase a c t i v i t y . Total nitrogen determinations were done on 1 gram weights o f the o r i g i n a l dried pulp.  Ten grams o f dried pulp  were extracted with 70 percent ethanol.  The alcohol extract  was analysed f o r sucrose and i n v e r t sugars,  insoluble n i t r o -  gen and 81arch-dextrin estimations were made on portions o f the alcohol extracted pulp. The v a l i d i t y o f the sampling method was tested by pulping the tissue o f f i v e beet roots, mixing i t w e l l , drawing f i v e separate 40 gram aliquots o f the pulp, and determining the phosphatase a c t i v i t y present i n each,  sucrose  was also analyzed f o r 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 a f t e r 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 t o t a l nitrogen decreased from the 1st to the 8th day and then increased a f t e r this time.  Insoluble nitrogen decreased from the 1st to the  8th day i n the decrowned and d e f o l i a t e d beets. gen results were generally inconclusive,  Insoluble n i t r o -  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 d e f o l i a t e d  and decrowned beets l o s t the highest dry weight percentages of sucrose.  The average fresh and dry weight percent of sucrose  i n the control beets was higher than the average percents i n the treated groups. The r e s u l t s f o r i n v e r t sugars indicated an increase i n the amount of reducing sugars following f o l i a r l o s s . percent of Invert sugars i n the control beets remained constant.  The fairly  The highest r i s e i n inverts was found i n the decrowned  beets, with the l e a s t r i s e i n the continually d e f o l i a t e d beets. The percent of starch-dextrins tended to decrease i n a l l treatments.  64*  A d e f i n i t e decrease i n percent of t o t a l 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 a c t i v i t y of phosphorylase decreased with time i n a l l treatments.  A c t i v i t y In the control beets  from the 11th day was considerably higher than i n the treated beets. phosphatase a c t i v i t y 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 a c t i v i t y increased s l i g h t l y towards the 4th day. then decreased u n t i l the 16th day. Beta-amylase a c t i v i t y was detected, but there were no apparent changes i n a c t i v i t y .  There was a tendency f o r  a c t i v i t y i n the treated beets to be lower than that of the controls i n the f i r s t experiment, but t h i s was not duplicated i n the second  experiment.  Catalase a c t i v i t y , on the basis o f monomolecular values, decreased with time a f t e r treatment,  on the basis  of T/C values, the a c t i v i t y increased i n the frozen beets from the 1st to the 11th day and then decreased.  A continual  decrease i n apparent a c t i v i t y was noted f o r the decrowned  65*  beets* A p o s i t i v e 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 i s suggested from the r e s u l t s *  In experiment  I I , invertase a c t i v i t y 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 a c t i v i t y  i n both defoliated groups was generally lower than i n the control beets* The r e s u l t s found i n this experiment were l a r g e l y 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 i n order to determine any differences i n 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-  7*  A oloser examination of invertase activity i n  pounds*  the beet root and i t s suggested relation to sucrose metabolism* 8*  studies to determine whioh type of sampling  method i s most accurate - including replication of treatments and analyses* 9*  studies with enzyme inhibitors i n relation to  the reduction of sugar losses, 10*  i o ^ o a & t a t © might be used*  studies involving certain growth substances,  as maleic hydrazide, and their efficiency i n reducing sugar losses following f o l i a r freezing*  11•  Research to f i n d the exact relationship between  sucrose losses i n p i l e storage and losses caused by f o l i a r freezing*  / 12.  .68.  LITERATURE CITED  (1)  PETO, P.H. E f f e c t of f r o s t on sugar content In beets, proceedings o f the American society of sugar Beet Techn o l o g i s t s . 108-111, 1952.  (2)  DOWLING, R.N. sugar Beet and Beet sugar. Brendon and Sons L t d . (Plymouth) 1928.  (3)  THE SUGAR REPINING INDUSTRY. Dominion Bureau of Stati s t i c s . Dept. of Trade and commerce, Canada. 1952.  (4)  PETO, P.H., SMITH, W.G. and LOW, F.R. E f f e c t s o f preharvest sprays of maleic hydrazide on sugar beets* proceedings of the American Society of sugar Beet Techn o l o g i s t s . 101-107, 1952.  (5)  STROHMER, P., BRIEM, H. and PALLADA, 0. Experiments on the d e f o l i a t i o n of sugar beets. M i t t . 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 o f dry mass and sugar i n the sugar beet root. Physiol, untersuch. zuckerube. Erste A r t i k e l Serie. ukrainischen Inst. Angew. Bot. sect, pflanzen physlol. 1934.  (7)  OPARIN, A.I. Storage of sugar i n the roots o f 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 i n t e n s i t y on growth, chemical composition and enzymic a c t i v i t i e s of sugar beets. Michigan A g r i c u l t u r a l Station Technioal B u l l e t i n 108 p. 44 1930.  (9)  The D i s t r i b u t i o n of Catalase i n the Sugar Beet. Zuckerlnd. Biehmen. 31. 201-17.  William  Z.  (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 f r e s h l y harvested beets. B u l l . Assoc. chim. sucr. D l s t . 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 y i e l d i n beets* Agr. Res* (U.S.), 54. No* 79* 639*654* 1937.  (15)  OPARIN, A.I. Metabolism i n sugar beet roots at low temperatures* storage o f beet root i n a frozen state* Compt* Rend* Acad* S c i . U.S.S.R. 2, 116*121* 1934*  (16)  BURMA, O.P. and MORTIMER, D.C. D i v i s i o n of Applied Biology, National Research Labs., Ottawa, Canada.* Biosynthesis o f uridine Diphosphate Glucose i n sugar Beet l e a f and i t s Possible Role i n sucrose synthesis*  (17)  HARRIS, F.S. The sugar Beet i n America. 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* CO. 477-478. 1951.  (20)  SUMNER, J.B., CHOU, T.C. and SEVER, A.T. phosphorylase of the Jack bean; i t s p u r i f i c a t i o n , estimation and properties* Arch* Bio chem. 26: 1-5* 1950*  (21)  GOTTSCHALK, R.G. Mi erode termination of acid phosphatase* Blochim* Biophys* Aota. 2; 582-589* 1948*  (22)  SUMNER, J.B. and SOMERS, G.F. Chemistry and methods of enzymes, second e d i t i o n . Academic press, New York. 1947.  (23)  SUMNER, J.B. and SOMERS, G.F. Laboratory experiments i n b i o l o g i c a l chemistry. Academic presa I n c . publ i s h e r s . New York, N.Y. 39-41. 1949.  (24)  SUMNER, J.B. and HOWELL, S.F. A method f o r determination of aaccharase a c t i v i t y , jour. B i o l . chem. 108, 51. 1935.  (25)  SOMOGYI, M* A method f o r the preparation o f blood f i l t r a t e s f o r the determination of sugar* jour* B i o l * Chem* 86, 655 (1930); 70, 599 (1926)*  (26)  ANALYSIS OF SUCROSE IN BEETS* A g r i c u l t u r a l chemists* 1946*  Jour*  Rural Science  MacMillan  Association o f O f f i c i a l  70. (27)  L00MIS. W.E. and SHULL, C.A. Methods In plant physiology. McGraw-Hill Book Company. New York. 1937.  (28)  SAILLARD, E . The reducing sugar content o f 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 e d i t i o n . 638639. 1952.  

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