CHANGES 8N CARBOHYDRASE ACTIVETES IN RED KEDNEY BEAN PLANTS TREATED WITH 2, 4-DICHLOROPHENOXYACETIC ACID Thesis for tho Deagwa of M. S. MKHIGAN STATE COLLEGE Wesiey Brock Needy W50 ,n- —-—_ 4 .— — __1—?~——' l . ! $ !. I. I i I 'lhis is to certih; that the f , n l ' i thesis cntitlml i ”ERNIE II: fl,‘FiVOFWWV‘3.0;.“ L.C';‘t\/"7'I‘Tz‘j.‘3 IN ”FD ' KIM‘IEX FERN F’LL‘T'CS TFFATLU .7713 l “fit-DW‘THOVT’PHE‘LI707W.Cl":II"7 LCTD ' I presented tn] l I Iesley Brock Keely I has been acveptnd towarcls fulfillmvnt at the requirements for ' . . -n”. —.- ————--$-- mu- 'lql-—-p pg blajul‘ ltrnft'smu‘ H" C O [’8th “.0er ,, 1, (:0 07169 CHANGES IN CARBOHYDRASE ACTIVITIES IN RED KIDNEY BEAN PLANTS TREATED WITH 2,LPDICHLOROPHENOXYACETIC ACID By weslqy Brock Neely A THESIS Submitted to the School of Graduate Studies of Michigan State College of.Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemistry 1950 Cit-WY um, ram»: 1 : 'h-Q g -.‘,' l ‘ ii“: ' I ACKNOILEDGMENT The author wishes to express his thanks to Professor C. D. Ball of the Biochemistry'Department and to Dr. H. 11. Sell of the Horticulture Department for suggestions and encouragement given during the work on this problem. Gratitude is also due to Dr. C. L. Hamner of the Horticulture Department for his kindness in preparing the plant material for analysis. w++++++ ++++++++ ++++++ ++++ ++ 4. TABLE OF CONTENTS Page Introduction 0 e o o o o o o e e e o o e e o e o 1 Historical e e e o e e e e e o o o e e o o o o o 3 Experimental Determination of alpha and beta awlase . . 7 Determination of Invertase . . . . . . . . 12 Determination of Pectin Methoxylase e e . . 1 Determination of Phosphorylase . . e . . . RBSUltS and DiBCHSSiOD e o e o e o e e e e e e e 16 Conclusion 0 e e e e o e e e o e o o e o o o e e 22 Bibliography 0 e o o o o e e o e o e e o o e e e 23 INTRODUCTION Increased knowledge in the field of growth-regulating substances has made it necessary to redefine terms (1). The term phytohormone or plant hormone is used in at least two different senses: a) in its restricted sense, as a naturally occurring correlation carrier, a sub- stance, according to van Overbeck, '---being produced in am one part of the organism, is transferred to another part and there influences a specific physiological process', and b) in a broad sense, as a 'growth regulating substance'. According to the first interpretation the con- venient term phytohornone cannot be applied to many synthetic substances which have not been shown to be plant products, yet have a physiological activity similar to that of natural substances. Thus naphthalenacetic acid would not be a hormone, whereas closely allied indoleacetic acid might be classified as such. Clearly the word phytohormone should be redefined as an organic substance which regulates physiological functions in plants. This definition includes synthetic as well as natural substances. Due to the wide publicity that plant growth regulators have received in the past few years a great deal of interest has been aroused as to what the actual effect these compounds have on plants. Plant hormones have become increasingly important in their commercial. application due to their physiological effects on various plants. Some are now used as weed killers, e.g., 2,h-dichlorophenotxyacetic acid, commonly called '2,h—D'. Others, such as naphthaleneacetic acid, are receiving more and more attention in preventing fruit drop in apples (2) and in develong parthenocarpic fruit (3). From the fact that these substances are needed in such small amounts and from the work done by -1- (O Sell et. al. (19) on the accumulation of protein and depletion of carbo- ‘hydrates in bean tissue treated with 2,héD, it was thought that this compound might act as an enzyme activater or inhibitor. Therefore, this present work was undertaken to discover what effect, if any, 2,hrD had on the carbohydrate enzyme system in the tissue of the Red Kidney Bean. HISTGIICAL The first mention to be found in the literature regarding the use of growth regulators as herbocides was the work done by Slade, Templaman, and Barton of England ()4). These workers as early as l9h0 discovered that a-naphthylacetic acid when applied at the rate of twenty-five pounds per acre to oats, 'weedy with field mustard', killed the mustard without injuring the oats. In November 19in these same authors (h) found that the substituted phenmqracetic and naphthoxyacetic acids were outstanding from the standpoint of selective herbicidal activity. Nutman, Thorton and Quastel (5) further tested 2,14-dichlorophenmqracetic and 2-methyl-h-chloro- phenoxyacetic acid to determine their merits as weed killers. In the United States credit for first suggesting the use of plant growth regulators as herbicides is given to E. J. Kraus of the University of Chicago, who, it is reported, suggested the idea as early as August 19141. In 191.2 Zimmerman and Hitchcock (6) published the results of their work with plant gowth regulators, and to them is generally given the credit for being the first investigators in the United States to danonstrate such physiological properties as cell elongation, root development, and partheno- carpy of substituted phenozqacetic acids. Kraus, Brown and Harmer (7) were the first to examine the histological changes in plants induced by growth regulators, however, they only used indoleacetic acid. Murray and Whiting (8) were the first to use 2 ,Lp-D. These authors applied the free acid and the amnonium, copper, calciun, and magnesium salts to the cut surface of decapitated Red Kidney beans, and at time intervals up to thirty days made histological examinations of tunor growths. For all five substances there was fundamental similarity in the tissues which responded and in the type of response induced. There were .30. e\ I\ '\ Ox characteristic differences in the location of the tumors, the speed of the response, and the time of maturation. The calcium and magnesiun salts caused the tumors to grow'near the surface, whereas the free acid and ammonium salt caused tumors considerably below the surface. The copper salt gave results intermediate between these two extremes. Modification of the pattern of leaves, flowers, and stems and the habit of growth is associated especially'with the influence of three groups of hormone-like compounds, naphthoxy, phenoxy and benzoic acids. The recent work of Burton (9) is one of the first attempts to determine what happens to the structure of tissue to bring about these altered forms. Using the bean leaflet as a test Object Burton worked out the normal structural developments and compared these with 2,hsD induced.modifications. The normal bean leaflet develops a lamina by the activity of a subepi- dermal marginal meristem which produced four internal layers of plate numistem. The adaxial (upper) of these layers develops into the palisade layer and the other three layers produce the spongy mesophyll. The veins are initiated‘oy division of rows of cells in the layer beneath the embryh onic palisade. ‘Iany intercellular spaces normally appear in the spongy tissue. Using three chloro substituted phenoxy acids Burton found that these were more or less specific for given structural variations from normal. For example, 2-chlorophenoxyacetic acid inhibited the formation of inter- cellular spaces in the spongy tissue, hpchlorophenoxyacetic acid inhibited the activity of the plate meristen. The 2,hsD brought about a progressive modification of all leaves developed after the application. The latter compound also caused various structural modifications similar to those produced'by both the other acids. Chlorenchymous tissue was usually 4‘. I. confined to the margin and the lack of chlorOphyll over the vascular tissue gave the veins a transparent appearance. Since plants are greatly modified in growth after treatment with physiologically active compounds, the question has frequently been raised whether these effects carry over into the second generation. Pridham.(10) found that seedlings of Red Kidney beans, from.parents sprayed with 2,hsD during the ripening of the pods showed a range of 2,hfiD symptoms in the Juvenile and mature foliage of the progeny plants. Virus-like, crisp dwarfing, serration, and.fusion of leaflets occurred to some degree in all seedlings. It is not entirely clear whether the holdover effects were due to substances adhering to the surface of the bean seed or whether they 'were due to substances actually stored in the embryo. I 'Willard (11) has recently reported a similar instance of abnormal effects being transmitted through the seed of corn grown.in single-cross fields. In this case seed from plants treated with 2,1t-D before tasseling was of low germination and the progeny was abnormal'in that they were slow growing and low yielding. The greatest 2,hsD effects occurred in progeny from treated plants showing the least ammount of injury in the field. In.reference to the importance of auxilarly substances added to growth.regu1ators to increase the activity, Lucas and Hammer (12) reported that Spanish onion juice increased.the activity of 2,héD'when applied to bean plants for inhibition of growth. These authors also tried the juice from.other plants, but found none as effective as Spanish onion juice. Spear and Thimann (1}) concluded that the action of onion juice is due to its content of sugar, phosphate and potassium ions. In addition many other substances have been reported which somewhat increase the killing -5- ,e effect of 2,h-D (1h, 15). In an effort to determine movement and translocation of growth sub- stances in plants Mitchell et. a1. (16) used a growth regulating sub- stance 2-iodo-3-nitrobenzoic acid (designated hereafter as INBA) labelled 'with.radioactive iodine 151. Bean plants were found to absorb and trans- locate this compound more readily than'barley plants. These authors considered the difference suggestive as to the relative susceptibility or resistance of species. in accmulation of INBA in the terminal bud of the bean plant caused a marked inhibition of growth. These authors concluded, however, that the difference in susceptibility of bean, oat and corn plants was probably due to the difference in the manner in which INBA reacts with the plant constituents in each case, rather than the amount of the chemical absorbed. It has been shown by Erickson and Mitchell (17, 18) that treatment of plants'with.2,hsD results in a reduction in.carbohydrate content and an accumulation of nitrogen. Sell et. al. (19) treated.Red Kidney‘bean plants as the first trifoliate leaf was expanding with an application of one drop of a 1000 p.p;m. solution of 2,héD to the base of the leaf. The authors then analysed the treated.and nontreated.plants for total nitrogen, ether extracts, carbohydrates and crude fiber. As compared to the controls their results showed that,2,heD treatment caused.an accumu- lation of protein and lipides whereas the reducing and nonpreducing sugars disappeared. The amount of starch, polysaccharides and crude fiber was decreased after 2, in application. EXPERIMENTAL Materials and Methods Samples of Tissue Seeds of the Red Kidney bean (Phaseolus vulgaris) were selected for uniformity of size and planted in four inch pots in the greenhouse. Each pot contained two plants, and two replications of one hundred plants each were used, from which samples of treated and nontreated plants were Obtained. Half of the pots (fifty pots) were treated by making an application of one drop (.05.) of a 1000 p.p.m. sclution of 2,hsD to the base of the blade of one of the prinmry'leaves. The treated and untreated plants were separately harvested six days after treatment.at the time the stem tissue had proliferated considerably but yet showed no signs of necrosis. The material was air dried in the dark and then segregated into the various parts. The hypocotyl, first internode and leaf petioles were grouped together as stem tissue and the leaves as leaf tissue. The roots were discarded. The material was ground fine enough to pass through the sixty mesh screen in the micro Wiley mill. The material was stored in glass jars at room temperature for a period of three to four weeks and used as DC eded 0 Analytical Methods Alpha and Beta Amylase Quadruplicate one gram samples of the treated and untreated stems and treated and untreated leaves were extracted separately with 100 ml. of water at 50°C. for one hour. The samples were then centrifuged, filtered and the enzyme activity was determined as described below. An alternative method of extraction with 100 ml. of 0.1 N sodiun bicarbonate in place of 100 ml. of water was also tried. The method of drying was also modified to the extent that fresh samples were subjected to freeze drying technique introduced by Stephen Djang of this laboratory. 0 The alpha and beta anvlase were determined by a slight modification of the methods described by Kneen and Sandstedt (20), Sandstedt, Kneen and Blish (21) and Blish and Sandstedt (22). Reagents l. The stock iodine solution was made up to contain 11 grams of iodine crystals and 22 grams of potassim iodide in 500 m1. of water. 2. Diluted iodine solution A. To 15 ml. of the stock solution 8 yams of potassium iodide were added and made up to 200 ml. with water. 3. Diluted iodine solution B. To 2 ml. of the stock solution, 20 grams of potassim iodide were added and made up to 500 ml. with water. )4. Dextrin solution contained 0.6 grams of Merck's Reagent dextrin made up to 1000 ml. with water. 5. A buffer of pH Luis-14.5 was prepared with 120 ml. of glacial acetic acid, 161; grams of anhydrous sodim acetate and diluted to 1000 ml. with water. 6. A beta amylase solution was made by extracting 1400 grams of hard wheat flour with 1000 ml. of water for five to six hours at roan tempera- ture, then centrifuged filtered through cotton and finally saturated with toluene. 7. A buffered alpha anwlodextrin solution was prepared from a sus- pension of 10 grams of Merck's Linter soluble starch, 25 ml. of acetic and sodium acetate buffer, 5 ml. of beta amylase solution. This solution was brought to 500 ml. with water and the: saturated with toluene. ~8- 8. A color standard was prepared by pipetting 5 ml. of iodine solution A into a comparative tube and adding 1 ml. of dextrin solution. This standard is usable for half a day. Determination p__f E322 M To 20 ml. of buffered alpha amylodextrin in a 50 ml. Ellenmeyer flask, 5 ml. of water were added and the mixture placed in a 50‘0. water bath. After temperature equilibriun 5 ml. of the solution to be tested were added. At appropriate time intervals 1 ml. of the reaction mixture was added to 5 ml. of iodine solution B in a comparative tube, then shaken and compared with the standard. Alpha amylase units are to be considered as the number of grams of soluble starch which, under the influence of an excess of beta amylase, are dextrinized by one gram of material in one hour at 50‘C. Reagents :2; Egg glass determination l. A buffer of pH hit-44.5 was prepared with 3 ml. of glacial acetic acid, h.l grams of anhydrous sodium acetate and then made up to 1000 ml. with water. 2. A one percent by weight sulfuric acid solution was prepared. 3. A 11/20 alkaline ferricyanide solution was prepared with 16.5 grams of pure dry potassitn ferricyanide, 22 grams of anhydrous sodim carbonate in one liter of so'thion. The solution was stored in a dark colored bottle. 14. A N/20 sodium thiosulfate solution was made up with redistilled water.fran dried 0. P. sodium thiosulfate pentahydrate. 5. An acetic acid reagent contained 200 ml. of glacial acetic, 70 grams of potassiua chloride, 20 grams of zinc sulfate heptamrdrate made up to one liter with water. ('- 6. A fifty percent by weight solution of potassium iodide was pre- pared, as well as a one percent by weight solution of soluble starch. 7. A buffered starch solution consisting of 10 grams of Merck's Linter soluble starch, 25 ml. of acetate buffer pH 14.14 was brought to 500 ml. with water and saturated with toluene. Determination _o_f_ Eta amylase To 20 ll. of the buffered starch solution in a 100 ml. Erlenmeyer flask was added sufficient water so that on subsequent addition of the enzyme solution the total volume was 50 ml. When the flasks and contents had cane to the temperature 30‘0. the enzyme was added and the hydrolysis allowed to proceed for exactly fifteen minutes. At the end of fifteen minutes the reaction was stopped by the addition of 20 ml. of the one percent sulfuric acid. To 2' ml. of the solution 10 ml. of N/20 ferricyanide solution was added and the tube immersed in a boiling water bath. After exactly 20 minutes the two was cooled in running water and the contents poured at once into a 125 m1. Erlenmeyer flask. The tube was then rinsed with 25 ml. of acetic acid reagent and the rinsings were added to the flask. One ml. of fifty percent potassium iodide solution, followed by 2 ml. of soluble starch were added with thorough mixing. Titration was then carried out with N/2O sodiun thiosulfate to the complete disappearance of the blue color. Beta amylase unit is defined as the number of grams of soluble starch converted to maltose by the beta amylase of one gram of material in one hour at 50‘0. gang Calculations £11113 glass The aliquot was equivalent to 0.05 grams of material or 0.Cil7 grams on a dry weight basis, and it dextrinized 20 ml. of starch (0.14 grams) in 16 minutes. The alpha amylase activity . 60 l was, therefore, equal to. .1; x 1‘5 x m . 51.9 units Beta agilase 1. The aliquot used was equivalent to 0.05 grams of material. 2. The ferricyanide reduced by 2 ml. aliquot is 6.1.7 m1. 10.1; milligrams of maltose is equivalent to 6.147 ml. of 0.05 N ferricyanide according to Blish and Sandstedt (21). 5. Maltose produced by 0.05 grams is, therefore, equal to 10.14 x 25 - 261 milligrams. (25 is the dilution factor) 14. Alpha dextrinization time for 0.05 grams material expressed in reciprocal units is '0 x 1 - .0575 units. .05 ‘13 From the tables prepared by Kneen and Sandstedt (20) the maltose that was produced due to alpha amylase by the nunber of units is equal to 11.87 milligrams. 5. Maltose due to beta amylase is, therefore, 261 - 11.87 - 2149.15 milligrams. 6. Starch converted to maltose is equal to 21.9.15 1: .95 Starch conversion in one hour 21+9eB X e95 3C 60 1000 15 Starch converted to maltose by beta amylase of one gram of plant material in one hour at 50'C. is equal to 249e13 x 095 _6_Q . ' , 1000 x .05 x .9141; x 15 33.14 units (.05 x .9141; ' grams of dry material used) .11- For invertase activity determinations a variety of extractions were used. The methods used were as described below (in each case all sanples after extraction were centrifuged and filtered). 1. One gram samples were extracted with 100 m1. of water at 50°C. for one hour. 2. One gram samples were extracted with 100 m1. of water at room temperature with gentle agitation for two hours. 3. One gram samples were extracted with 50 ml. of water for one how at 55°C. It. One gram samples were extracted with 100 ml. of water for two hours at 50'C. I 5. One gram samples were extracted with 100 ml. of water for ten minutes in the Waring Blender at room temperature. 6. One gram samples were extracted with 0.1 N acetate buffer at pH 14.5 for ninety minutes at 50‘C. Determination of Invertase Activity by a Modification of Willstatters Method (25). Forty grams of Pfanstiehl sucrose were dissolved in a 0.1 N acetate buffer of pH h.5 and diluted to 250 ml. with the buffer. The solution was mixed and the Optical rotation was determined in the polariscOpe. The flask containing the remaining solution was placed in a thermostat bath at 50‘C. (he milliliter of the solution to be tested was introduced into a 50 ml. volumetric flask and diluted to the mark with the buffered sucrose solution. At the end of a definite period of time 0.2 grams of sodium carbonate were added, shaken and a reading taken in the polariscOpe. Readings were taken at the end of 50, 60 and 90 minutes. The rotations were then plotted against time in minutes on coordi- nate paper and the number of minutes extrapolated to reach zero rotation. This is the time value of invertase. One invertase unit has a time value of one. Determination of Pectin Methoxylase Activity - by method of Z. I. Kertesz Extraction of’material for Pectin methoxylase activity was carried out as follows. Two gram samples of the tissue were extracted with 50 ml. of water for one hour at 50‘C. The samples were then centrifuged filtered and aliquots used for activity measurementstas described below. Approximately one percent by weight citrus pectin (Eastman) solution was made up and 50 m1. measured into a 125 ml. Erlenmeyer flask. Using a Beckman pH meter the solution was adjusted to pH 6.2 using 0.1 N sodium hydroxide. A.5 ml. aliquot of the enzyme solutiOn was added to the citrus pectin solution maintained at 50‘C. in a water bath, noting the time of the addition of the enzyme. Sodium hydroxide was added from time to time to keep the pH at 6.2. The reaction was run for thirty minutes noting the volume of standard alkali required to maintain the pH constant. The Pectin methoxylase unit is definednas the number of milligrams of methoxyl split off“by one gram.of material in thirty minutes at 50‘C. _S_a__le_e. Calculatiog l. O.h.m1. of 0.1 N sodium hydroxide were required t0»maintain pH at 6.2 during the thirty minute time interval. 2. There were 0.0L. grams of plant material in 1 ml. of the enzyme. solution used. Since 5 ml. of solution were used, there were 0.20 grams of plant material. 5. For every equivalent of base there were 51 milligrams of methoxyl split off. Therefore, the activity is calculated as follows: 0.1+ x 0.1 le .20 . milligrams of methoxyl split off by one gram of material in thirty minutes at 30.00 Determination of Phosphorylase Activity - by the method of Green and Stunpf (2 5) Two grams of plant material were extracted with 50 ml. of water at 58‘C. for one hour. The material was then centrifuged, filtered and aliquots were analyzed for enzyme activity as described below. The test mixture consisted of 1.8 m1. of enzyme solution, 0.5 m1. of 0.5 M citrate buffer at pH 6.0, 0.2 ml. of five percent soluble starch and 1 ml. of 0.1 M glucose-l-phosphate giving a final volume of about 3.5 ml. The reaction was allowed to proceed for ten minutes at 58‘C. and then stopped by addition of 5 ml. of five percent trichloroacetic acid and 2 ml. of 2.5 percent ammonia molybdate. The mixture was filtered - and 0.5 ml. of molybdate and 1 m1. of reducing agent (1,1l-aminonaphthol- sulfonic acid) were added and the solution diluted to 25 ml. The blue color was allowed to develop for five minutes at 58'C. and then read in a Klett Summerson Photoelectric Colorimeter. This method for phosphorus determination was described by Fiske and Subs-row (26). 1 A blank determination on each sample was made by adding the tri- chloroacetic acid imediately after the enzyme. The rest of the pro- cedure was the same as described above. The instrument was zeroed by making a blank containing 2.5 ml. of molybdate, 1 m1. of reducing agent and diluting to 25 ml. It was allowed to stand‘for five minutes at 58°C. before setting the instrument at zero. A standard phosphate solution was prepared by dissolving 0.5509 grams of mono potassium phOSphate in water, 10 m1. of 10 N sulfuric acid was added and the solution diluted to one liter. (1 m1. - 0.08 milligrams phOSphorus) This solution was made to calibrate the instrument. To 1 ml. of the standard phosphate solution, 2.5 ml. of molybdate solution and 1 ml. of reducing agent were added, then diluted to 25 m1. and allowed to stand for five minutes at 58°C. A reading was then made in the calorimeter. (he unit of phosphorylase activity has been defined as the amount of enzyme which catalyzes the liberation of 0.1 mg. of inorganic phosphorus fran glucose-hphOSphate in three minutes at 58°C. at a pH of 6.0. £3221: Calculations Reading for the standard . 7.8 .08 mg. Phosphorus - 7.8 Control stems after enzymatic hydrolysis 1.75 Control stems - blank reading _2_63 Reading due to acticm of enzyme 210 Phosphorus liberated in ten minutes w - 2.12 milligrams Phosphorus liberated in three minutes - .636 milligrams Therefore, one gram of material has 3-3173 - 8.91.. units of phosphorylase activity RESULTS AND DISCUSSION The results from separate samples for both the alpha and the beta amylase determination are shown in Table 1. Table 1, Effect of’2,héD on.Alpha and Beta Amylase Activity in the Stems and Leaves of Red Kidney Bean Plants. Stems Leaves Enzyme Sample nontreated treated nontreated treated 1 29.91 11.65 0 0 alpha 2 31.90 11.90 0 0 amylase 3 33.32 5.02 0 0 h 33.20 5.25 0 0 Avg. 31083 14-095 0 O 1 38.70 21.15 25.87 27.91 beta 2 33 01-1-0 25.99 26e20 26e90 amylase 3 35e60 26022 27.62 28 0314 1. 58.60 22.51. 25.62 26.31. Avg. 36c 58 23.93 26033 27037 (All units expressed on a dry weight basis.) These results represent individual runs on the two enzymes. Although there was a variation in the results for each separate determination, the discrepancy was not enough to change the over-all picture, namely, that 2,héD inhibited the activity of the alpha and beta amylase in the stems of the Red Kidney bean. The beta amylase activity in the leaves of the plant showed a very slight increase in the treated tissue, but this slight activation is not significant due to the experimental errors involved in the determination. An over titration of 0.1 m1. will change the activity by one unit. The variation in securing the original sample of the plant material must also be taken into consideration when interpreting these results. -16- These results, namely, that 2,héD inhibits the activity of alpha and beta amylase of the stems, which would result in a decreased con- version of the starches to degradation products, tend to confirm the work done by Sell et. al. (19). These workers in their analysis of the stems of Red Kidney Beans showed that application of'2,h+D caused a disappearance of the reducing and non-reducing sugars. The apparent lack of effect of 2,h~D on the alpha and beta amylase of the leaves presents a pr0b1em. It would be interesting to run analyses for reducing and non-reducing sugars on the treated and uns treated leaf tissue to see whether or not 2,héD'would cause a depletion of these compounds similar to that observed by Sell in the stems of the plant. Erlich and Burkert (27) showed that extraction of malt with a 0.1 N sodium bicarbonate solution increased the alpha amylase activity, but did not significantly alter the beta amylase activity. This was tried on bean tissue and the results confirmed the above Observations as noted in.Table II. The relative activities, however, remained un- changed as compared with the data in Table I. Table II. Effect of the 0.1 N Sodium Bicarbonate Extraction on the Alpha and Beta Amylase of the Treated and Nontreated Leaf and Stem.Tissue of the Bean Stems Leaves Enzyme nontreated treated nontreated treated alpha 56.1. 5.66 0 0 amylase 57.8 5.60 0 0 beta 57.0 2h.0 25.57 27.20 amylase 57.82 23.8 26.20 261.2. -17. puns-o- a..—..- 1" _ -~..—.o- .- ---_, ~-- “g... Table III shows the effect of one eXperiment that freeze drying had on the activities of the amylases. The results are very interesting but the work should have been repeated on different lots of plants before any definite conclusions could be made. Table III. Effect of Freeze Drying on the Alpha and Beta Amylase of Bean Tissue. Stems Leaves Enzyme nontreated treated nontreated treated alpha amylase 6 0 0 0 beta amylase 25.9 6.02 13.95 D4 It is interesting to observe that these values, although signifi- cantly lower than the previous values, are still of the same relative order. This observed effect might be explained by the fact that since freeze drying has the least effect on the tissues chemically, these determinations might be the actual activity for the enzymes. Air drying the samples in a heated room, on the other hand, might increase the enzyme activity above these actual values. Despite all the different attempts to extract invertase from the tissue, no indication of invertase activity by any of the methods used could be found in either the control or treated leaves and stems. This 'would seem to indicate that the breakdown of sucrose to glucose and fructose by invertase is lacking in the tissue of the young bean plant. Table IV shows the results of the pectin methoxylase activity studies from separate samples of both treated and nontreated stems and leaves of the bean plants. .45- Eéh;e IV. Effect of 2,}4-D on the Pectin Methaxylase Activity ,in the Stems and Leaves of the Red Kidney Bean. Stems Leaves Enzyme Sample nontreated treated nontreated treated 1 6.2 15.81 301 7090 Pectin 2 7.1411 16.10 2.79 8.05 Methoquase 3 8 .96 16.27 3 .10 8 .20 h 7.90 16.11 2.914 8.05 The other pectin enzymes were not tested for, but if the ultimate breakdown of protopectin to methanol and galacturonic acid is a series of equilibriun reactions, then the increase in pectin methoxylase activity would likely be associated with an increase in breakdown of the protopectin in the cell walls. This is exactly the phenomena observed in histological studies in young plants that have been treated with 2,14-D. Separate samples of treated and nontreated stem and leaf tissue of the bean plant were tested for phosphorylase activity and the results are shown in Table V. Table V, Effect of 2 ,h—D on the Phosphorylase Activity of the Stems and Leaves of the Red Kidney Bean. Stems Leaves Enzyme Sample nontreated treated nontreated treated 1 *9.32 5.55 5.00 0.1128 PhOSphory- 2 8.76 5.91 3.98 0.725 1888 3 8.98 5.68 2078 00771 l. 3.16 0.5m *Units are expressed as phosphorylase units. -19.. It was thought that the liberation of phosphate might be due to a phosphatase enzyme. Although no glucose-lpphosphatase has been reported in the literature, the possibility still existed. Therefore, it was decided to ‘ determine the ‘ r reducing sub- stances before and after enzymatic action. If the reducing substances increased then it would be due to the liberation of glucose from the glucose-l-phosphate by a phosphatase. However, if the reducing sub- stances remained constant then the liberation of phosphate would be due to a phosphorylase converting glucose-l- phosphate to the corres- ponding glucose polymer. The Folin and'Wu.method for the determination of reducing sugars 'was employed (28, 29). This method depends on the reduction of phos- phomolybdic acid by the cuprous oxide to produce a blue color which is read in a photoelectric calorimeter. The cuprous oxide is produced from any reducing substance which will convert the cupric ion in Benedict's reagent to cuprous oxide. The results of this experiment as given in Table VI. showed that there was no increase in reducing substances due to the action of the enzyme solution in either the treated or control leaves and stems. Therefore, the liberation of phosphate must be due to a phosphorylating enzyme 0 Table VI 0 Results of Determining the Reducing Substances Before and After Enzyme Action on Separate Samples of the Bean Tissue. Stems Leaves Sample nontreated treated nontreated treated Before After Before After Before After Before After 1 .2562 .2526 .626 .6282; .382 .3886 .14260 .1202 2 .2602 .2628 .6211 .6252 .38h .3832 .1428 .8268 3 .2681. .26h8 .6140 .8401. .3812 .3816 1.302 .1298 h .2652 .2660 .8411. .6072 .3888 .3862 .8320 1.322 ‘(Readings are given in Optical Density) -20- All readings were made on the Iflett Summerson photoelectric colori- meter where the scale reding is related to the optical density by the . 2R D - optical density formula D ' 1000 R - reading on the instrument From Beers' laws the decrease in the intensity of the transmitted light is proportional to the concentration of the solute in solution where the depth of the cell is a constant, or in mathematical form %—I- . 4% IO - incident light 0 Integration yields us the following expression lnI . - 3 KG 131% . optical density 0 o Therefore, D .- -KC Thus optical density is directly proportional to the concentration of the solution. These results on the phosphorylase activity help to confirm the previous results, namely, that application of 2,11-D tends to alter a very definite step in carbomrdrate metabolism and thus may be one of the reasons for accumulation of proteins and lipids in the treated plant. This is exactly what Sell et. a1. (19) Observed in their chem- ical analysis of treated and nontreated stem tissue of the Red Kidney Bean plant. CONCLUSIONS 1. The alpha amylase activity was inhibited by application of 2,hAD in the stem.tissue, whereas no activity was observed in either the treated or nontreated leaf tissue. 2. The beta amylase activity was decreased in the treated stem tissue as compared to the nontreated. No change in activity was observed in the leaf tissue. 3. Invertase was apparently absent in.both the treated.and untreated stem and leaf tissue. 14.. Pectin Methoxylase activity was increased «by . a the appli- cation of 2,h~D in both the stems and leaves of the bean plant. 5. 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