SOMS EFFECTS OF DIFFERENT FERTILIZER TREATMENTS ON THE DIURNAL AND SEASONAL CHANGES IN THE SUGAR CONTENT OF THE SAP AND TISSUE OF POTATO PLANTS *>y RALPH CHASE COLE A THESIS PRESENTED TO TEE FACULTY OF MICHIGAN STATE COLLEGE OF AGRICULTURE AND APPLIED SCIENCE IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY East Lansing 19 3 1 ProQuest Number: 10008705 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10008705 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 06- 1346 Acknowledgement The writer is greatly indebted to Doctor M. M, McCool, formerly of this institution, for assistance in planning this work, and to Doctor C* E, Millar, of the Soils Department, also Doctor R, R, Hibbard, of the Botany Department, for criticisms and suggestions in the preparation of the manuscript. CONTENTS Introduction ......... Historical 1 .... »...... 2 Experimental Outline ...... S Weather Conditions .......... 9 Methods of Sampling ............................... 11 Preparation of Samples .................. 11 Method of Analyses ................................ l4 Discussions and Conclusions IS ....................... 26 Summary....... Literature C i t e d ........... 27 SOME EFFECTS OF DIFFERENT FERTILIZER TREATMENTS ON THE DIURNAL AND SEASONAL CHANGES IN THE SUGAR CONTENT OF THE SAP AND TISSUE OF POTATO PLANTS. INTRODUCTION Because of the large increases in yields resulting from the application of fertilizer salts to fields that ordinarily produce plants which from all appearances are normal and vigorous, much interest has been shown in studies of the growth habits, nutrient requirements, and physiological processes of plants. The problems involved in the search for a more intimate understand­ ing of the way plants respond to stimulations of any hind are many and have attracted workers from many fields of endeavor. Much work has been done on external factors such as climate and the physical and chemical nature of the soil. Studies of the plants themselves considering their anatony, ecology, habits, method of reproduction and chemical analyses of the various parts of plants have been made and from all of this work much valuable information has been obtained. alyses Among the many chemical an­ made of plant tissues, carbohydrate analysis of various parts of plants and the study of the rates of movement of these constituents is one of thdjmost interesting phases of all chemical analyses. The study of carbo­ hydrate variations is a difficult one because of the large number of carbo­ hydrates and the wide range of fluctuations in concentrations from hour to hour. Most of the work done in the past has been directed towards an attempt to determine how carbon assimilation takes place, and how the products of carbon -2- assimilation are transported from the leaves to the parts of the plant in which they are utilized or stored for future use. Although some stud­ ies have been made showing the tendencies of different carbohydrates to vary in concentration in different parts of plants throughout the day, the need for a more complete knowledge of these changes, and how the ef­ fect of certain cultural conditions affect the normal growth of plants was felt. This work was planned so as to find out more about the variation in the carbohydrate concentrations resulting from applications of fertil­ izer salts in amounts that have proven practical in of potatoes. The potato plant increasing the yields was selected because the author had been working with fertilizer requirements for potatoes for several years and also, because of the habits of the plant in storing carbohydrates, it lends itself very well to this type of investigation. HISTORICAL Sachs(1) in 1862 first proved that the production of starch in the chlorophyll granule depends upon the action of light and that the starch formed during the hours of sunlight is wholly or partially redissolved from the leaf during the night to supply the demands of the growing points of plants. This discovery by Sachs aroused great interest in studies of carbon assimilation. Probably the first theory on the problem of carbon assimilation was put forth by Liebig ^ in 18*4-3» acids were the. intermediate products. which he considered that organic This theory was not ba-sed upon ex­ perimental evidence and found little or no support. A theory that has -3- directly influenced so much experimental work is the formaldehyde theory, in which it is claimed that formaldehyde is formed from water and carbon dioxide in the green part of the leaf* condensed to sugar* Baeyer (3) in 1S70 This formaldehyde is immediately The formaldehyde theory was first proposed by A. is widely accepted at. the present time. The first sugar formed according to this theory is grape sugar, d - glucose, which is later enolized to fruit sugar - fructose and from glucose and fructose, sucrose is formed. From sucrose and the simpler sugars thejmore complex carbohydrates of storage such as starches, hemicelluloses and celluloses are formed. Most of the early work along this line of investigation consisted of attempts to prove this theory. The methods used, however, were almost entirely qualitative and as such are now subject to much criticism. In 1S93 Brown and Morris (^) who were among the first to use quan­ titative methods in these studies came to the conclusion that sucrose was the primary sugar of photosynthesis. They found sucrose to be much more abundant in the leaf than starch and the method in which it fluctuated caused them to draw this conclusion. They considered dextrose and fructose to be the products of hydrolysis of sucrose rather than those materials from which it was synthesized. Parkin ^5) working with the snowdrop, found that at different periods during the day even when the leaves of the plant were covered with black paper, the concentration of the hexoses remained about constant while the concentration of sucrose varied with the amount of sunlight and temperature of the day. He concluded from this that sucrose was the primary sugar of photosynthesis. -14- fC\ Davis, Daish & Sawyer working with translocation of car- bohydrates in the leaves and steins of the mangold, and Davis & Sawyer (7) working with translocation of carbohydrates in the potato plant, found that during early stages of growth the content of sucrose was always greater than that of the hexose in the leaf, but the reverse was true in the stems. They claimed this to be proof that in the leaf sucrose is the primary sugar of photosynthesis and is converted into the hexoses for means of transportation. Their reports contain an excellent review of the literature on the subject of the primary sugars of photosynthesis up to their time. Dixon & Mason (S) made microchemical examinations of the assim- ulating cells of a number of plants and found that there was a consider­ able concentration of hexoses in the chloroplas-ts or in the protoplasm immediately surrounding them. Sucrose on the other hand was concentrated in the vacuoles and invertase was held apart from it in the protoplasm. They concluded from this that the hexoses are first formed from formal­ dehyde in the chloroplast and where their concentration reaches a certain limit condensation into sucrose due to invertase or some other saccharigenic enzyme takes place. Priestly took the rather unusual view that sucrose is not a pro­ duct of photosynthesis, but a decomposition product of protoplasm. He based this view principally upon the fact that chemically sucrose had never been synthesized from fructose and dextrose, and no enzyme had been found in the plant to which could be ascribed this function. In his paper he gives an excellent review of the literature or the subject especially with reference to papers - concluding the hexoses, especially dextrose to -5- be the primary sugar of photosynthesis. Clements (10) in making carbohydrate studies of the leaves of several plants concluded that dextrose is the primary sugar of photosyn­ thesis primarily because of the way it varies in concentration in plants In comparison to the variations of sucrose. The subject of carbohydrate - nitrogen ratio in plant tissue has been of great interest to horticulturists. A consideration of the purpose for growing the plant has been of prime importance in this field. In cases where the purpose is to produce vegetative growth, a different carbohydrate nitrogen ratio, is required than when the plant is grown primarily for reproductive growth. Among the outstanding researches in this line is that of Kraus & Kraybill (ll) . Working with the tomato plant they found: 1. Though, there be present an abundance of moisture and mineral nutrients including nitrates, yet without the available car­ bohydrate supply, vegetation is weakened and plants are not fruitful. 2. An abundance of moisture and mineral nutrients, especially, nitrates, coupled with an available carbohydrate supply makes for increased vegetation, barrenness and sterility. 3. A relative decrease in the nitrates in proportion to the carbohydrates makes for an accumulation of the latter, and also for fruitfulness, fertility and lessened vegetation. H. A further reduction of nitrates without Inhibiting a possible increase in carbohydrates, makes for a suppression both of vegetation and fruitfulness. -6- (12) Reid working with, basal and upper cuttings of tomato plants found those high in nitrogen furnished favorable conditions for shoot growth while those high in carbohydrates appeared to furnish better con­ ditions for root growth. H o o k e r w o r k i n g on the changes in chemical composition of (l^+) apple spurs, Murneek' ' on the nitrogen and carbohydrate relations in (organs of) apple bearing spurs, Harvey & Murneek (1*5) on the carbohydrate and nitrogen relations in apple spurs all show that the carbohydrate and nitrogen ratio bears an important part in determining whether or not a spur will be fruitful or barren. These workers all came to the same conclusions as Kracts & Kraybill. A very noteworthy piece of work on carbohydrate studies was done by Mason and Maskell (16) in studying the translocation of carbohydrates in the leaf, stem, bark and ball of cotton plants. They were able to assertain the rate of movements in specialized parts of the plant. They also found that there was a lag in the cycle of maximum carbohydrates from leaves to bark. Miller (17) working with the leaves of corn and sorghams found that in most cases the non-reducing sugars were in excess of the reducing sugars. The non-reducing sugars increased markedly during the day and decreased during the night while the reducing sugars as a rule showed very little increase and the amounts present at the different periods of the day were irregular. Janssen & Bartholemeu (IS) found in working with tomatoes grown in sand cultures that there seemed to be an optimum potassium concentration which was condusive to the normal assimilation of carbohydrate compounds, -7- and above or 'below which, assimilation was reduced. This optimum relation was not found. The same investigators. (19) also found hy working with a variety of field crops "both on sand and water cultures, that maximum concentration of carbohydrates were produced at concentrations of 2 to 3 PPm po­ tassium, while applications of from 50 to U50 pounds of potassium chloride per acre to the soil on which the same crops were grown showed no corre­ lation between the amount of potassium added to the soil and the carbo­ hydrate content of the plants. They also found reciprocal relations between the potassium and nitrogen content of the plants when grown on sand or water cultures, but not with those grown in the field. /20 \ Clements found reciprocal relations for nitrogen and carbo­ hydrates in water cultures using field peas. However, he found the highest percentages of carbohydrates in the cultures that received the most cal­ cium nitrate and the highest percentages of nitrogen in the cultures re­ ceiving high pi*oportions of potassium di-hydrogen phosphate. Woo (21) working with Amaranthus Retroflexus, a plant that is capable of storing large quantities of nitrogen found there was a reciprocal rela­ tion between carbohydrates and nitrogen, both in the leaves and the stems. This reciprocal relation was especially noticeable between insoluble protein and carbohydrates doubtless, because the carbohydrates are utilized in the formation of proteins. Hartwell (22) found that a deficiency of available potassium in the soil was usually accompanied by an accumulation of starch in potato vines. He also found that many different factors which correlated in each case with retarded growth, were found to be associated with an accumulation -s- of starch in the above ground portion of plants. Experimental Outl ine. The object of this experiment was to study the diurnal and sea­ sonal variations in the sugar content of both leaves and stalks of the potato plant, for the purpose of noting any differences that might occur due to the application of commercial fertilizers under field conditions, and to see if these differences could be correlated with yields. The diagram below gives an outline of the way the field from which the samples were gathered was laid out. 2,A0/)S 4—16—0 tK.00 0-16-3 4-16-S 4-16-12 « 0-16-3 4-i6-*o * 4-16-12 0 4^16-3 S The treatments were replicated four times, the other two replications joining the above on the south. The samples were taken from the check plot, the 4-16-0, 0-16-3 and 4-16-S of the north series and the other three series were left for taking yields. The 4-16-12 plot was not sanpled because previous experiments had shown that there was no differ­ ence in yields from plots fertilized with 4-16-12 and 4-16-3, and it -9- was considered advisable to keep the number of samples as low as pos­ sible. The H-16-12 treatment was included in order to further compare it with H-16-S from a standpoint of increasing the yields. The field used for this experiment was located about a mile and a half from the laboratory. The soil was Hillsdale sandy loam, which is one of the principle morranic soils of south central Michigan and is well adopted to the growing of potatoes. The fertilizers applied were made from (HHl^gSO^ as the nitrogen carrier Uo$ superphosphate as the phosphor­ ous carrier and muriate of potash as the potassium carrier. and broadcast by hand at the rate of 750 pounds per acre. It was mixed The fertilizer was worked into the soil with a spike-tooth drag before the potatoes were planted. Weather Conditions The season of 1930 was an extremely dry one, the yields of pota­ toes being consequently greatly reduced. The yields obtained showed no amounted to differences due to treatment and only lyfceisbsi from 90 to 100 bushels per acre while in years of normal rainfall f rom 200 to 300 bushels per acre could be expected. The plots were sampled on July 23, August 12 and September 4,and over this period only O.5S of an inch of rain, fell while the normal for this period is from four to five inches. The first samples were taken when the blossoms were forming, the second as the plants began to set tubers, and the third sampling when the plants began to mature. An attempt was made to take samples only on a clear day, and samp­ lings were never taken unless the day previous had been clear. This pre­ caution was taken in order that the normal production and utilization _ -10te* of carbohydrates might be going on within the plant at the time when the samples were taken. The following data from the United States Wea­ ther Bureau at East Lansing give; the meteorological conditions on the dates of sampling and for two days previous to each sampling. Date Min. Temp. Max. Temp. July 21 67 89 22 63 82 23 53 August 10 Character of Day Sunshine per cent Precipitation in inches 68 .04 tl 72 0 33 clear 99 0 52 73 clear 96 0 11 U5 73 it 94 0 12 U5 73 ii 100 0 September 2 68 37 cloudy 23 .07 3 5k 82 clear 95 0 k 46 79 100 0 pt. cloudy 1! ii In spite of the dry weather the plants seemed to be perfectly normal and unhurt during the first two samplings. The plants showed that the drought was hurting them by the time the third sampling was made. They were fresh during the night, but during the day the top leaves curled slightly and the plants looked wilted. When the first samples were taken the plants in the fertilized plots were much larger than those in the check rows. This difference was not so noticeable when the second samples were taken showing that the larger plants were not growing as fast as the smaller ones. Even in normal years much greater differences between fer- -11- tilized plots are noticed in the earlier part of the season than can he detected later on. By September 4 when the third samples were taken the plants showed clearly that they were injured by the prolonged dry spell. The leaves curled up and the plants looked slightly wilted from the middle of the day on until after dark, although they looked fresh in the morning. Ho differences could be seen in size between the plants in the check rows and on the fertilized plots. MSthods of Sampling. At each sampling seven sets of sables were taken. Starting at midnight samples were taken every four hours through midnight of the follow­ ing day. In taking samples the whole hill was selected, tops being cut­ off about two inches above the ground. fill a twenty pound capacity paper bag. Sufficient hills were taken to In the first sampling six hills were taken from each fertilized plot, but eight hills were required from the check plot to give . sufficient material. It was only necessary to take four hills from each fertilized plot and five from the eheck plot for the second sampling whereas four hills gave sufficient material for each from all treatments in the third sampling. Preparation of Samples. The samples thus obtained were rushed to the laboratory immediately after cutting and disposed of as quickly as possible. each treatment was separated into leaves and stalks. The cutting from Part of each was then chopped up as fine as could be conveniently cut with a paper cutter and 50 gms of each was put in separate pint jars containing *JQO cc of boil­ -12- ing alcohol to which had been added a little calcium carbonate to neu­ tralize any acid that might be found in the plant. The use of the paper cutter permitted the leaves to be cut in strips about l / k of an inch wide while the stalks were cut in sections about half an inch in length. The jars were set on the steam bath to boil for one hour in order to kill the enzymes immediately after the tissue had been added to them. The remainder of the sample of leaves and stalks was ground separately in a food chopper and then from 100 grams of each the juice was pressed out according to the method described by Sayre and Morris (23) amount of juice pressed out from the leaves exceed 50 cc. . ITever did the It was mea­ sured and all of it poured into bottles containing 200 cc of boiling alcohol which had a little calcium carbonate added. The juice from 100 grams of ground up stalks usually amounted to from 60 to 10 cc, 50 cc of which was the leaves. pipetted off and preserved in the same manner as the sap from The samples thus obtained were also put on the steam bath and boiled for one hour. All of the samples from both tissue and sap were tightly sealed after heating for one hour and stored away to be anal­ yzed later. In order to have the samples comparable in all respects, the same order of handling was maintained in the laboratory for all samples so that the time between cutting in the field and the killing of the enzymes would be about the same for each treatment throughout the period of sampling. The time between cutting the samples in the field and getting all samples into boiling alcohol was just about one hour. Although it would have been better to have had less time between cutting in the field and getting them in hot alcohol, there were so many samples that they could not be carefully -13- handled in any shorter time. Samples of both, sap and tissue were preserved because it was considered possible that additional information might be gained by an analysis of the sap. An attempt was made to determine the sugar content of sap with­ out the use of preserving agents. In this attempt, the diluted juice was treated with neutral lead acetate to clearify the solution. The precipitate caused by the lead acetate was thrown down by centrifuging and the clear liquid poured off into another centrifuge tube. The lead was precipitated from solution by using powdered sodium oxalate and the precipitate removed by centrifuging. The supernatant liquid was poured off into a dry container and an aliquot taken made up to volume ready for analysis. The solutions prepared in this manner and determination made immediately had about the same reducing power as samples that were pre­ served in alcohol. Their reducing power did not remain constant and at the end of three or four days they exhibited very little if any reducing power. As it was impossible to make the determinations immediately after sampling it was necessary to find some way of maintaining the reducing power of the samples constant. The method of preserving in alcohol was known to be satisfactory, but before determinations could be made, it was necessary to change the samples from an alcohol solution to a water solution and a search was made for a more convenient method. Ripperton^?^ working with carbohydrate metabolism in the edible canna found that when he took the expressed sap and clearified it with lead then removed the lead and made up to volume the reducing power of these samples changed rapidly, and he could not use this method. He -1*J- next tried using formaldehyde and found that by adding formaldehyde, the percent of total sugars remainded constant, but the sucrose continued to invert* He found the method of preserving in alcohol quite satis­ factory. further searches for a preservative so as not to necessitate the use of alcohol showed that the use of a half gram of sodium fluoride main­ tained the reducing power of samples constant for at least ten days. Other samples in which the lead acetate was used as a preservative and was not removed until just previous to making the determination proved effective in maintaining a constant reducing power in the solutions. It is usually considered that the presence of lead in fructose solutions will decompose the fructose and thus change the reducing power. These solutions treated with lead acetate and left to stand did not change their reducing power as they must have been rather low in fructose* Although the use of either sodium fluoride or lead acetate indi­ cated a possiblenBthod of preserving samples, before any further investi­ gations could be made it was necessary to sample the field, and hence both the sap and the tissues were preserved in alcohol. Methods of Analyses* The samples that were preserved in alcohol were filtered and made up to volume* From these, aliquots were taken and the alcohol removed by distillation under reduced pressure using Classion flasks* The solutions in the Classion flasks were boiled down to about JOcc, then washed out into 100 cc volumetric flasks with hot water. The volumetric flasks were then cooled down rapidly by setting them in running tap water for several minutes. -15- then allowed to stand until cooled to room temperature. At this time j.” , , the .contents zney were made up to volume, shaken well and/poured into 100 cc centri­ fuge tubes, which previously had about one tenth of a gram neutral lead acetate added. The solutions were allowed to- stand for fifteen or twenty minutes to permit all of the colloidal material to be precipitated. The precipitate formed was thrown to the bottom of the tube into a sticky mass by centrifuging. The supernatant liquid was poured off into another dry centrifuge tube and powdered sodium oxalate added to precipitate all the lead. A second centrifuging threw down the lead oxalate precipitate and the supernatant liquid was decanted into a dry beaker. An aliquot of this liquid was made up to 100 cc volume. The samples thus prepared were thoroughly shaken, 50 cc of the solution put in a 300 cc Erlenmeyer flask for reducing sugar determina­ tion and the remaining 50 cc put into another 300°c flask to which had been added 5 grams of citric acid crystals used to hydrolyze all sucrose. The samples for sucrose analysis were next taken, covered with 100 cc beakers and put on a gas hot plate and permitted to boil for ten to fifteen minutes to hydrolyze the sucrose. The citric acid was next neutralized with concentrated sodium hydroxide to a pink color with phenolphthalein. The reducing power of both the sucrose samples and non-reducing (25) sugars was determined by the Shaffer and Hartman ^ method, using a standard solution of sodium thio-sulphate to titrate the excess iodine from a known amount of potassium iodide-iodate added to the reduced cop­ per solutions. Twelve cc of 8U sulphuric acid were immediately added to the solution after the iodide-iodate had been added, shaken for a few seconds until all the cuprous oxides had been dissolved, then 20 cc of -16- saturated potassium oxalate was added and the solution titrated with standard sodium thio-sulphate solution. As the end point of the titra­ tion was nearly reached, 5 cc of starch solution was introduced as an indicator and the titration completed. Due to the buffer effect of the citrate ion in the sucrose samples it was necessary to add an additional 5 cc of the sulphuric acid in the titration to bring the samples to the necessary pH* The committee on methods for the society of plant physiology^^ object to the use of the Shaffer and Hartman methods on the ground that for various tissues it is not always possible to get a sharp end point in the final titration. In this work, by using the recommended 15 cc of 5 normal sulphuric acid the end point was not always sharp, but by in­ creasing the strength of the acid a clearly defined end point could always be obtained. It appeared, at lea.st, with the tissue used in this work, that there is a rather narrow range of pH in which a sharp end point can be obtained with this titration. If an excess of acid is added a white precipitate forms in the solution and the resulting titration values are too low, while if the pH is too high, the end point is indistinct and the results are unreliable. The addition of extra acid, in the case of sucrose hydrolyzed by citric acid, to correct for the buffer effect of the citrate ion indicates the necessity of getting a proper degree of acidity in the solutions before a clear end point can be observed in the titration. With a little practice one can usually tell by the color of the solutions as the acid is being added, just how much acid to use and if working with tissue from only one kind of plant, the correct acidity can soon be found and no further difficulty. wi H noted. -17- If the difficulty lies in obtaining a definite pH value before titrating, it is easy to understand why all tissue cannot be handled alike because it is easy to conceive of different tissues exerting dif­ ferent buffer effect on solutions, which would necessarily have to be corrected for before sharp e^d points could be obtained. There are a number of other methods of determining the amount of reduced copper (26, 27), but the Shaffer & Hartman method has a distinct advantage over most of them in that it is not necessary in this method to filter off the products of the reaction before the titration can be made for the determination of the reduced copper. An excellent review of the literature on methods is given in the former reference. The method of reducing the copper described by Quesumbing & Thomas (28), was employed in this work because it was more convenient in handling a number of samples at one time than is the Munson & Walker (29) method. Although the Quesumbing & Thomas method of reduction was used, the Munson & Walker tables were used for calculating the amount of sugar from the amount of copper reduced, because the Munson & Walker tables are so much more complete* The curves on Figure I show the variations between the two tables within the range of copper obtained in this work. The differ­ ence between the two is so slight that the error due to interpolation chf the smaller tables of Quesumbing and Thomas would be greater in many cases than the difference between the two tables. In working out the method of analysis it was desirable to work out a plan whereby a set of eight samples, which is the number of samples that were taken at each cutting could be handled in a day. This was desirable in order to have the method of handling for all samples as nearly alike js o & jx s Q jo e w to w n -1S- as possible even to the time "between starting a determination and the final titration. In adopting such a method it was necessary to choose certain procedures, such as distillation under reduced pressure rather than evaporation from a steam hath to get rid of the alcohol, and the use of citric acid hydrolysis for sucrose rather than inversion by inver­ tase, because these proceedures were a little faster and fitted in better with the scheme of analysis. The use of the centrifuge in removing precipitates from leading and deleading was much more rapid and conven­ ient than filtering for this purpose. In using any more rapid method, a careful check up was always made in order to see that accuracy was not sacrificed by the use of a more rapid method. Discussions and Conclusions The data are presented in the form of graphs, which seems to be the most effective way of showing diurnal . variations. All of the free reducing sugars are reported as glucose and all material hydrolyzed with ten per cent citric acid solution is reported as sucrose. Series A, content figures 2-7 shows the diurnal variations in the carbohydrate/of all the samples, due to the effect of the different fertilizer treatments. All figures are given as percentage of green weight. The curves in figures 2-7 follow almost the same course, showing that if there is any variation due to the different fertilizer treatments the variations are indeed small. Some irregularities are seen in some of the curves, but a careful observation shows that these irregularities are not consistant with any one treatment, but all of which show some irregularities. The data show that under the conditions of this experiment -19- no differences due to differences in fertilizer treatment can be expected. This is consistant with the work of Janssen and Bartholemeu (l9) who worked with a number of field crops, in the field and in sand and water cultures. In growing the crops in the field they applied constant amounts of nitrogen and phosphorus, and potassium varying in amounts from nothing up to U50 pounds of potassium chloride per acre. In this case no variation in the carbohydrate content of the tissue due to different amounts of potash added was observed. When the same plants were grown in water cultures and sand cultures they found a maximum car­ bohydrate production in solutions containing from two to three parts per million of potassiu#. C lements^found in water cultures that a high carbohydrate content in the tissues of field peas was correlated with a low nitrogen content. The point of maximum carbohydrate production in the works of both Clements and Janssen and Bartholemeu are not always the points of maximum production or maximum growth. Kraus & Kraybill (ll} working with sand cultures found definite relations between nitrogen and carbohydrate content in the tissue of to­ mato plants. They were able to establish from these relationships some fact pertaining to the functions of different parts of the plant with reference to vegetation or reproduction. It seems that results obtained with sand and water cultures under conditions in which the concentrations of the solutions in contact with the roots of plants are kept constant cannot be easily duplicated in f ield plot work. The difference in all probability rests in the fact that soil -20- conditions are dynamic and not under control. There are wide variations in moisture supply and an extreme variability in the soil solution in the field. Both the per cent of moisture and the concentration of the soil solution are affected by so many factors under field conditions. Svery rain dilutes the soil solution, the losses of moisture by transpiration tends to concentrate it while the thermal movement of water in the soil (30) has its effect on the soil solution. Iyon & Buckman give a good des­ cription of the dynamic character of the soil solution and its relation to the moisture contents of the soil. Factors of this nature appear to exert so much more influence on the plants that the effect of added ferti­ lizer salts although sufficient to cause marked influences in yields sel­ dom show any influence on the concentrations of carbohydrates in plants. This suggests that it may be possible by using water culture or sand cul­ tures to adjust differences of concentrations of different nutrient elements between narrow enough limits so that distinct differences in growth of the plants may be observed without any noticeable differences in the carbo­ hydrate content of the tissues or sap of the plant. A further examination of the curves in series A indicates closer relationship between the curves of figures 2 and 3 than those of figures 4-7. The first mentioned curves are those from the first sampling on July 23, when there was not as much sugar found in the plants as there was later. It is only natural to expect then, that when there is a larger amount of sugar in the plant any variations that may oc­ cur due to various factors, will cause greater differences than will occur under the same conditions in plants that have a much lower sugar content. -21- The curves in 130111 the second and third samplings show greater irregular­ ities in data from the stalks than in that from the leaves when both tissue and sap analyses are considered. this variation. There seems to be no apparent reason for An observation of the alcohol extracts indicates from a prior reasoning that the data from the leaves would be more irregular because of the larger amounts of coloring matter and the greater difficulty in clari­ fying the solutions. With the exception of set (a) at the top of figure three, all of the curves in figures two and three are almost horizontal.The curves for glucose in the second andthird samplings, figures ^-7* a re horizontal or nearly so, while those for the sucrose seem to indicate clearly a minimum from 4 A.M. to 8 A.M. and a maximum b etween noon and U P.M. In the second sampling a minimum occurs at 8 A.M., but in the third sampling the minimum may be either at k A.M. or at 8 A.M. A comparison of the amounts of hexoses reported as glucose, and of sucrose (figures 7-i3) shows that in all three samplings the amount of the former is in excess of the latter in both the leaf and stalk samples. 2?hese findings are directly opposite to the findings of Davis and Sawyer^) t wk0 found that samples taken on July l6 and 17, 191^» showed sucrose to be greatly in excess of hexoses at this time* They did find, however, that the percentages of hexoses seemed to remain constant throughout the day and night whereas the sucrose content showed a steady rise from 6 A.M. until about 2 P.M., at which time there was a gradual decrease until the mini­ mum was reached at U A.M. In the stalks of the same sampling they found the hexoses to be greatly in excess of the and sucrose running almost parallel sucrose, thecurves for the hexoses and showing a slow but steady rise from 6 A.M. until sunset and then dropping off to a minimum about 2 hours before sunrise. -22- Although the general trend of the curves for hoth sucrose and glucose in this study are similar to those of Davis and Sawyer, the amounts of sucrose as compared to glucose in the leaves are directly (17) opposite. Miller , working on the carbohydrate variations in corn and sorghum leaves found in practically all cases that the percentages of non-reducing sugars were much greater than those of the reducing sugars in both types of plants. C l e m e n t s r e s u l t s , on sunflowers, potatoes and soy beans do not conform with those of Davis & Sawyer or Miller. He found on July 6 and 7 that in the leaves of potatoes the curves of both simple sugars and sucrose are almost horizontal and the amounts of simple sugars are greatly in excess of sucrose. The curves for both sunflowers and soy beans on the same dates show the curves for both simple sugars and sucrose also to be about horizontal, but for these plants the amounts of sucrose are almost equal to those of glucose. Samples of potato leaves taken August 11 and 12,and 26 and 27 also show the amount of simple sugars to be in excess of sucrose, although, the percentages of both have in­ creased over those from the July 6 and 7 samplings. In both of the later samplings the curves for the simple sugars are extremely irregular. This is also true for the later samplings of sunflowers and soy beans. There seems to be no definite cycle similar to that noted in the work of Davis an* Sawyer and Miller, and also in the second and third samplings of this present work. It seems logical to expect a rather definite cycle showing periods of maximum and minimum concentrations in sugars throughout the day. The work of parkin, on the carbohydrate in the snowh&rop and Mason & Haskell (l6) on transport studies of carbohydrate in the cotton plants also seem to point out that there is a definite cycle in the sugar content of leaves of plants* In the work of Mason & Maskel and that of Parkin (5) samples were taken at six hour intervals, in this investigation at four hour inter­ vals, and in that of Davis & Sawyer and of Miller at two hour intervals* Clements took his samples at intervals of one hour. Due to the great irregu­ larities in his curves Clements concluded that intervals of one hour were too great so he sampled sunflowers on September 15, 1926* at intervals of 10 minutes between the hours of 11 A.M. and 2 P.M. In this case his curves were much smoother, but still there were rather large variations in the concentration of simple sugars between the 10 minute intervals. A comparison of the percentages of sugars between the sap and tissue of the same samples (figures 8 to 13) shows that although the curves do not lie as close together as might be expected, yet the general trend of the curves are the same. The irregularities in the curves are not consistent and no factor of conversion can be obtained to correct for the difference between the two. The variations in the first sampling seem to be just as wide as those in the second and third sanplings. In a number of cases the comparison curves run just as close together as those of Sayre and Morris ^3) who made studies of the sugar content in the blades, sheaths and stems of corn* The third set of graphs, Series C, figures lU to 22, compare the amounts of the different sugars in the samples at different times of sampling. As might be expected, the concentration seems to be generally higher the later the sanpling, at least the range of concentration is — 24— greater for the older plants. The later samplings also show a much greater irregularity in concentrations than is shown in the first sampl­ ings. There seem to he no variations due to different fertilizer treat­ ments that are brought out by this manner of arranging the curves. A careful examination of the curves presented in this work as well as those presented by others who have worked on diurnal variations in carbohydrate concentrations in plants, reveals a great fluctuation in all forms of carbohydrates studied . These fluctuations vary to a greater extent in older plants than in the younger ones. The curves for the later samples of sunflowers, potatoes and soy beans, as shown oy Clements (10) are extremely irregular. Even the analysis of sunflowers at 10 minute intervals by the same author shows that the simple sugars vary from about .63$ at 11:40 A.M. to about .44$ at 11:50 A.M., and back up to about .62$ again at 12:00 .M* . while the minimum content of simple sugars for the whole period of three hours is .44$ and the maximum about .73$. It seems almost impossible to conceive of carbohydrate concen­ trations varying as greatly as they do in this work and the work of Clements. Erom a careful examination of a number of methods for determining reducing and non-reducing sugars it appears that anyone is sufficiently accurate to give reliable results at the time the analyses are made. If, however, the samples had changed any in their carbohydrate content between the time of sampling and the time the analyses were made, it would appear more than probable that they would have occurred between the time the samples were cut and the enzymes killed. This interval of time was much greater -25- in this work than that of Davis and S a w y e r o r Clements. Clements seems to have been especially careful in getting his samples into 95$ alcohol not more than ten minutes after the samples were cut. It is entirely possible that the heating of the material previous to the time of completely killing the enzymes causes great changes in the carbo­ hydrate content. There may be some other -factor that is causing changes in the samples. extracts of plants found • thus far not observed (30 Webster in studying alcoholic that amino nitrogen decreased in amount during the storage period frem-the™t-3^e=-4he~seiaplee are stored. He found no regularity in the decrease, but when potassium nitrate was added to sam­ ples of spinach at the time they were preserved, they showed greater de­ creases in amino nitrogen than spinach without potassium nitrate. Young alfalfa plants which are high in nitrates also had large decreases in 0/ aimp nitrogen. He suggested that the amino acids are probably converted to ammonia and a ketone according to the following equation: fi CH-UEs 5 ' COOH / H3 c ~ 0 * 38H, \ CHO n He further suggested that if the equation represents the true reaction taking place, then it is reasonable to expect an increase in the reducing power of the solutions and an increase in ammonia. It is not unreasonable to believe that if the work of Webster gives a true indication of changes in amino nitrogen content of extracts when stored, it is possible that other changes are also taking place, -26- and it may give an indication as to why the curves in work of this kind are so very irregular. There seems to he a need for further studies on the methods of preserving plant tissues for various types of analyses and especially those types of analysis dealing with or­ ganic materials such as carbohydrates and proteins. SUIliABY 1. Analyses showing diurnal and seasonal variations in the sugar content of both the sap and tissue of the potato plant are given. 5* Under the conditions of this experiment no differences are seen in the sugar content due to the difference in fertilizer treatment, 3. The curves for both glucose and sucrose seem to run almost horizontal for the early samples, whereas the leaves of the later sam­ ples show a minimum concentration of sucrose from U A.II. to g A.M., and a maximum concentration from noon to U P.M. Glucose curves seem to run horizontal in all samples. H. The percentages of glucose are greater than those of sucrose in all samples of leaves as well as stalks. 5 . The concentrations of both sucrose and glucose are higher in the later samples than in the early samples. 6. Greater irregularities are noticed in the analyses of stalks than in the analyses of the leaves. 7 . Comparisons between the sugar analyses of tissue and sap of the same plants show the curves to be very much alike and yet not so similar as^-/ might be expected. S. An opinion as to why carbohydrate analyses show such irregularities in variations as noted in this and other work is presented. -27- Literature Cited 1. Sacks, J., 1862: IJber den Emfluss des Lichtes auf die Bildung des Amylums in den Chlorophyllpornerm, Bot. Zietung 20: 3^5-373 2. Liebig, J., 18^3: ,fChemistry in its Application to Agriculture and Physiology'1, 3rd. ill. Philadelphia, Peterson. 3* Baeyer, A., 1870: Ueber die Wasserentziehung "^nd ihre Bedentung fur das Pflauzenleben und die Gahung, Ber. d.d. Chem. Ges. 3: 63- 7S 4. Brown, H. T., and Morris, G.H., A contribution to the chemistry and physiology of foliage leaves. Jour. Chem. Soc. 63: 60^-677» 1S93* 5- Parkin, J. 6. Davis, W.A., Daish, A.J., and Sawyer, G.C., Studies of the formation and translocation in plants I The carbohydrates of the mangold leaf. Jour. Agr. Sci.7: 255~326, 1916 7. Davis, W.A. and Sawyer, G.C., Studies of the formation and trans­ location of carbohydrates in plants III. The carbohydrates of the leaf and leaf stalk of the potato. The mechanism of the degradation of starch in the leaf. Jour. Agr. Sci. 7; 352-^SU, 1916. The carbohydrates of the foliage leaves of the snowdrop and their bearing of the first sugars of synthesis. Bio Chem. Jour. 6: 1-^7, 1912 8 . Dixon, H.H., and Mason, T.G., The primary sugar of pholosynthesis. Hature, ^7: l60. 1916 9 . Priestly, J. H., The first sugar of pholosynthesis and the role of cane sugar in the plant. Hew Phytol. 23: 255-265 192k. 10. Clements, H.F., Haurly variations in carbohydrate content of leaves and petioles. Bot. Gaz. 89 L 2^1-272. 1930 11. Kraus, H.J., and Kraybill, H.R. , Vegetation and reproduction with special reference to the tomato. Oregon, Agr. Sxpt. Sta. Bui. 1^9. 191S 12. Hied, 1.1.1., Growth of tomato cuttings in relation to stored carbo­ hydrates and nitrogen as compounds. Am. Jour, of Bot. 13: 5^6 - 57^- 1926. -28- 13* Hooker, H.D., Seasonal changes in the chemical composition of apple spurs. Mo. He s. Bui. UO. 1920 1*1. Murneek, A.E., Hitrogen and carbohydrate distribution in organs of apple bearing spurs. Mo. Res. Bui. 119, 132& 15* Harvey, E.M., and Murneek, A.E., Relation of carbohydrates and nitrogen to the behavior of apple spurs. Ore. Agr. Expt. Bui., 176. 1921 16. Mason, T.G. and Maskill, E.J. Studies on the transport of car­ bohydrates in the cotton plant. I A study of diurnal variations in the carbohydrates of leaf bark and wood and of the effect of ringing. Ann. of Bot. 1+2: 190-253. 17. Mill er, E.C., IS. Daily variations of the carbohydrates in the leaves of corn and the sorghums. Jour. Agr. Res. 27: 7S5-S0S. 1921* Janssen, G. and Bartholemeu, R.F. The translocation of potassium in tomato plants ard its relation to their carbo­ hydrate and nitrogen distribution. Jour. Agr. Res. 3S: 1+1+7-465. 1929 19. Janssen, G. and Bartholemeu, R.F. Influence of potash concentration in the culture medium on the production of carbo­ hydrates in plants. Jour. Agr. Res. 1+0: 2U3-261. 1930 20. Clements, H.F., 21. Woo, M.L., Plant nutrition studies in relation to the trian­ gular system of water cultures. Plant Phys. 3: 441-458, 1928. Chemical constituents of Amaranthus Retroflexus. Bot. Gaz. 68: 313~3^5* 3-919 22. Hartwell, B.L., Starch congestion accompanying certainfectors which retard plant growth. R. I Agr. Expt. Sta Bui. 149 1918. 23. Sayre, J.D. and Morris, V.H. Use of expressed sap in physiologic studies of corn, Plant Phys. 6; 139““l4S. 1931 24. Repperton, J.C., Carbohydrate melabolism and its relation to growth in edible conna. Hawaii Agr. Expt. Sta. Bui. 56, 1927 25. Shaffer, E.A. and Hartman, A.P. The codometric determination of copper and its use in sugar analysis. Jour, biol Chem. 1+5: 3^5“390. 1920 -29- 26. Loomis, W.E. et al. 27. Dorlittle, et al. 2S. 551© Determination of soluble carbohydrates Section III of the report of the committee on methods of chemical analysis of the American Society of Plant Physiologists. Plant Phys. 2: 195-204. 1927 (editing committee) Official and Tentative methods of analysis of the Association of Official Agricultural Chemists. Quesumbing, E.A., and Thomas, A.W. Conditions affecting the quanti­ tative determination of reducing sugars by Fehlings solutions. Jour. Amer. Chem. Soc. 43* 1503-1526. 1921 29. Munson, L.S. and Walker, P.E. methods* 1906. Hie unification of reducing sugar Jour. Chem. (Amer) Soc. 2Sj 663-6S6 30. Lyon, T.L. and Buckman, H.O. The nature and property of soils (pp. 233-240). Revised edition 1929. The MacMillan Company, Hew York. 311 Webster, J.U. Effects of storage on alcoholic extracts of plant tissues. Plant Phys. 4; l4l-l44, 1929 -30- The following tables of data give the detailed analyses from which the curves were drawn. All figures denote percentages of sugars based on the green weight of the plant or the sap, as the case may be. -31- lst Sampling Leaves 7-23-30 HQ Fertilizer______________________ 4-16-0 Fertilizer Glucose Gluco se Suoose Sucrose Glucose Glucose Sucrose Sucrose Sap Tissue . Sap 4 Tissue Tissue Sap ,Tissue Sap Time of Mid night 4;00 A.M. 3:00 A.M. Ho on 5:00 P.M. S :00 P.M. Mid night . . .200 •3^7 .402 .115 .161 ,351 .443 .560 .176 .4i6 .439 .662 .229 .416 .627 .675 .246 ,493 .310 .3S1 .261 .430 .701 .421 .230 .450 .515 .■?21 .313 _____ .649 .620 .246 .153 .393 .541 -199 .365 .617 .600 .119 .264 .392 .613 .195 .129 .424 M3 .156 .l4o .235 .337 .143 Ms .223 1st Sampling Leaves Time of S&mpliBg Mid night 4;00 A.M. 3:00 A.M. Hoon 4:00 P.M. 3:00 P.M. Mid Hight 0-16-3 Fertilizer Glucose Glucose Sucrose Sucrose Sap Sap tissue tissue .234 - ..-, 4-16-3 Fertilizer Glucose Glucose Sucro se Sucrose tissue Sap Sap tissue .142 .177 .274.. .504 ,l40 .126 ^255.. . ,359. . .533 .249 .232 .362 .537 .225 .446 .523 .409 .130 _____ •3I_1. _,353 .401 .299 .532 .536 .3_93_ _ .317 _ .415 .671 .442 .203 .442 .645 •32_6___ .17^ .369 .512 .272 .222 .513 _ ,5QI_ .176 .231 .432 •44_9_ . ,133 .251 .209 .462 .152 .111 .203 .529 .296 .090 .413 _ ___________ . . -32- Time of 'Glucose Sampling .sap Mid night .413 ftOO A.M. ..,3.13 S;00 A.M. .646 Ho on 1:00 P.M. 8:00 P.Mti Mid night 1st Sampling Stalks zer Glucose Sucrose' Sucrose tissue .... sap tissue 4-16-0 Fertillzer Glucose Glucose’Sucrose 1Sucrose tissue sap tissue sap .334 0 .156 .371 .313 .106 .119 .191 .189 .318 .397 .256 .110 .167 .478 .143 .202 .561 .345 .066 .217 .581 .567 .244 .159 .631 „ 7”23“30 .200 ...697 .471 .195 .297 .714 .489 .182 .251 .691 .567 .246 .529 .701 .558 .261 .485 •397 .694 .io4 .160 .613 .507 .044 .132 1st Sampling Stems 0-16-8 Fertilizer __ 4—16—8 Fertilizer r -— -n Sucrose Glucose Glucose Sucrose Sucrose Time of Glucose Glucose Sucrose sap tissue tissue sap tissue tissue sap Sampling sap Mid .481 .321 .150 night .249 .176 __.131_. .131 ____ .257 4:00 .362 . .296 _ .182 .171 •535 ,253 _ .106 A.M. •237___ 8:60 .081 *238 .068 .483 .274 .187 ,331 A.M. .505 Hoon 4:00 P.M. .483 .398 .201 .222 .438 .437 .336 .131 .235 .489 .642 .393 .118 .297___ .548 .265 •379 .195 .104 .293 .368 .186 .166 _ .390... .143 .272 ....09S .108 .238 •339 .238 .175 8:00 P.M. Mid night -33- 2nd Sampling Leaves No fertilizer Time of Glucose Glucose Sucrose Sucrose Sampling sap tissue sap tissue Mid night •536 .296 .294 -=507 4; 00 A.M. .482 .547 •599 .497 8:00 A.M. _ .524 .299 Uoon fc;00 P.M. 8:00 P.M. Mid night 8-12-30 .533 .599 .326 .378 .370 .539 .454 .428 .474 .611 .341 .195 -5U7 .761 .426 .343 .489 .792 .540 .422 .643 .761 .640 .507 .523 .719 .663 .515 .559 .529 .407 .564 .738 .536 .469 .529 •177 .515 .U5I+ .525 0-16-8 fertilizer Time of Glucose 5€Q?S>ling sap Mid .3+82 night .303 .537 .126 •475 4“l6?8 Fertilizer Glucose sap Glucose Sucrose Sucrose tissue sap tissue •195. .3+52 .529 .391 . .371 .564 .709 .298 'Boon .530 .857 .300.. . .329___ .785 .497 .3+71 .746 .509 .258. _ _ .438 . . .523 .433 .115 .. . .323+ .538 .463 ...... . Glucose Sucrose Sucrose tissue sap tissue .200 -.329 .60S .473+ .427 ._706 .768 . .249. .286 ...5. 69;. .701 ._=65I._. -^522 _ .751 .357____ 0 • 0 ^ £ .537 8:00 A.M. 4:00 P.M. 8:00 P.M. Mid night 4-16-0 fertilizer Glucose Sucrose Sucrose tissue tissue sap Glucose sap ..... .„ ...36.7 .. .561 ___ *558.... _ . j a . .675.... .574 . ,JJ2 .682 .409 .315 .551 .269 •577 ... . -34- 2nd Sampling Stalks Kb ^fertilizer___________ T-- .■ Time of Glucose Glucose Sucrose Sucrose .Sampling sap tissue sap tissue Mid night .602 .4ss .350 .,157 i+:00 A.M. .734 .286 j SSlL ., _ .335 3:00 A.M. .661 .101+ .244 ..,513 Uoon .594 .714 .236 . ,3.4s 4:00 P.M.____ ..-•312 .402 _ .507 _ .ste 8:00 P.M. 1.054 .m .165 .447 Mid night .00 .935 .357 .393 0—16—8 Fertilizer tfime of Glucose Glucose Sampling sap tissue Mid night .532 •415. 5:00 .697 a .m . .631 8:00 .446 A.M. .357 . noon 4*00 P.M. 8:00 P.M. Mid ni^at . Sucrose Sucrose sap tissue 8-12-30 4-16-0 Fertilizer Glucose Glucose^ Sucrose 'Sucrose sap tissue tissue sap .492 .650 .251 .281 .768 .811 .306 .286 .95 .682 .00 .251 .605 .649 .226 .481 .686 .185 .286 .663 .104 .262 .00 .325 .793 1.01 .299 .559 Glucose Glucose Sucrose .. -v. sap tissue sap .180 .134 .328 ,315_ .245.... .699 .00 .294 .868 .368 . Sucrose tissue .114 .219 .79.2 . .222 .294 .559 .247 .308 .328 _ ,357 .600 .501 _ .421 .299 .572 .422 .704 .431 .380 .447 .730 . .504 .650 .563 .201 .433 .716 .-..,329. .207 .417 •905 •599 .00 •3S5 .632 .407 .093 .313 _*316.. . .280 -35- 3rd Sampling Leaves 9-4-30 Ho Fertilizer____________ 4-16-0 fertilizer Time of Glucose Glucose Sucrose Sucrose Glucose Glucose Sucro se Sucrose Sampling sap tissue sap tissue tissue sap -!-« sap tissue Mid night .496 ^539 .441 .400 .396 .291 .439 .338 4:00 A.M. .810 .212 __.5S5, .314 .247 .499 .923 .263 8:00 .344 A,M. .700 .400 .497 .309 .0843 .203 .631 HoonO 4:00 P.M. SjOO P.-':. Mid night Time of Sampling Mid night 4:00 A.M. 0 • O»* ^51 SO 0-16-8 Fertilizer Time of Glucose Glucose Sampling sap tissue Mid night ..>555 .619 4*00 ,862 A.M. .757 S;0Q .424 .788 A.M. Noon .520 .518.. . 5*00 .932 . .438 P.M. 8*00 .937 .731 P.M. Mid 1.032 l.04l night *1-16-8 Fertilizer Sucrose Sucrose tissue sap Glucose Glucose tissue sap .290 .289 .611 >325 .522 .326 .1+02 .835.. ..1.119 .358 .427 .301 .1+02 .656 .631 .349 .229 .Ul5 .31+5 .441 .4ll .635 >375 .713 .61+2 .. ,158..... .1.^01 .32U .321 .. 1.056.. 1.313 •2l+3 .21+6 _ .827 1.313 - Sucrose Sucrose sap tissue .656 ..•363 ...... >3.89.. .237 .446 .1+1+5 l u Sa/ipw/yg Sa p - glu co se- L eaves 0 -/6- « 4 -/ 6-8 1 st . S a m PUA/6 T i s s u e - Glucose- Leaves J ST. Sa/IPUM* Sap- Glucose- S talks 1 st, Sampua/g Tissue- Gifi/c©sE.-Stalks 1 st S a * \ p u a j & S u c r o s e .- S a p - L e a k e S Jtr Swpling SuCRose-T»SSDt-L£AVES 1 st S a/ a p u w & S u c r o s e - S a p - St a c k s 1 s t S a m p c ja /g S u c ro s e - T is s o t - S talks 4-/e-a -OC check, ' 4 -ie -o o -/e -g MiOtf/GHr FIC. 3 2-no S/V'-iPUA/fc G t u c o s c - S a p -£ je a v £ S £/V0 SajAPUA/6 G»i.ucose- T issue ,- L eaves 2.W6 SA/KPC/NCG lucose. - S a p - S tacks /.t> £*e> 8aaapc/w& fic i; c .o s £ - -Tissue -Stacks 8 MtDMKZtJT SaMPWWG, I S u c r o s e - S a p - L e a ves CHECK A-16-o io-ie-8 Z>N6 SAP*PiWA/&j S u c r o s e - T is s u e -; L eaves 2 , a ii > i S a m pua «&: S u c r o s e - S a p - 'St a c k s — >$ s 4 -1 6 - g J o - (6 -8 &/U& SAMPi-/Af& S u c r o s e - T is s u e - S t a c k s Mtt» '/GUT F/6,5 S e r ie s A a 3fto Sa/aplin& .8 & LU CO SE - S a p - LEA VES .7 4-/6-8 .6 4--/6-0 O' 16-8 CHECK .4 .3 .1 ■o » t3 3aD Sl\A\KUWC> G>loc o s t - Tj s s o e L eaves /» M 4 -/6 - 8 4-ifi-o CHECK .3 .2 •o *4 e 3rd Sa^iac/a/g G l u c o s e -Sa p - S t alks tz M 0 - 16-8 lo .9 4-/6-fl •8 CHECK .7 *6 .S' .4 .3 • I •o MtOtkteur g f)"i v /?at FIG. 6 * Asoo/v ¥ PM t PM fintoNtGur Glucose - / - T is s u e - S t a l k s PASf 3RD SAMPLING Sucrose.-Sap-Leaves 3/10 Sampling. Sucrose- T is s u e - L eaves X 3 rd Sa m p u n g , S u c r o s e - S ap - S talks 4 - is - 0 CHECK, 3ro 3amrung S u c ro s e - T is s u e -S ta lk s -> U j Sampling - Leave!s glucose-tissue. GLUEOSE-5AP Su c r o se -S A P 5UCRO.SE- T ISSU E G LU C O S E.- T I S S U E G LU C O SE' SAP . S u c r o s e .- S A P ^ S U C R O S E - T IS S U E -.0 GLUCOSE- TISSUE s GLUCO SE - S A P i s g c R o s t-S A P *• SUCROSE - T I S S U E GLUCOSE.-TJSSU£ *S G L U C O S E - SAP S U C R O S E - T I5 5 U C jV O O A f S U C R O S E ' TISSUE. Su c r o s e - sap G LUCOSE - SA P Al.U£ojy^.msUE. Su c r o s e - t i s s u e Su c r o s e - 5A P GLUCOSE-TI55UE 1 GUJCOSE- SAP ,1 S u c r o s e .- SAP ■i SU CR O S E-TIS SU E G L u c o S C - T IS S U E &LU C.OSE - S A P SUCROSE - SAP SUCROSE.- T is s u e FIG. 9 fcrtD S a MiPLIMG - L fav S eries B CHECK j & U )tO S E -T IS S U E ■==?iotRoStrTWSVE — 4 (*LU C O S E -S A P — &LOCOSEj^TISSUEi ^ SUCROSE-tissue.’ ■&IUCOSE- SAP i&(,yt0S£r.SAP__. « S U C R O S E -TISS U E ' iC U C O S E ' TISSUE sucrose - T is s u e G iU tO S E - SAP LUCOSE-T/S5 U E &LUCOSE- SAP - SUCROSE- SAP S U C R O S E -T fS S U E M/O/tr. G H 7- */ F \& . tz A/OVAf 4 *>*? M!DAf/6Jj7- Series B * GLUCOSE-S^R * GLUCOSE- T'MUE ■Nr--SOCRost -TISSUE S O C R o & E - 5A> ■.©■ y Su c r o s e - Sa p -St ACK5 4-16-Q 0 S ucrose -Tissue - S talks 4 -1 6 -0 £ nci S a ^ pli n o 3 rei S a m p l i n g - G lu c o s e .- 3 a .p - IL e a v e s 0 - 16-6 G l u c o s e .- T i s s u e - o-(6-8 -O Q lu c o s e - S a p - STACKS 0-/6-e 5 •o Gl u c o s e - T issue .-'S t a l k s £>- 1 6 - 8 ■» 1 s t S a Fi6. la m p l in g S e r ie s C, L e a v e s -S u c ro s e -5 a P 0 - / 6- 8 /s rr S a m p l in g Zno Sampling L e a v e r - S ue. a & se. - t >s 5 u e O-Vfi-B -o Stalks -Sucrose- Sap 0-J6-8 Stalks - Sucrose-Tissue o - 1 6 -6 Is r Fie. id Sa m p l in g E.AVE5 i£n b•SAhKlAJk 0- Glucose-TissuE-4 - j e - a £ms SAin?i.iw& AST_J?A&£iJiJS. Gl, u c 06 e - -5taLKS 4-16-6 &LWC.05E-T»sswe-5TAUKS 43Ab 5 A M P U N & 2./4b 5AKPLINC 1st SAhKlMk floCHoSE-S/SP-LEAVE6 4 -/6 -6 S u c r o s e - T rs s o E - L e A v e s 4-16-8 Znt, SA*l>UA/&. Sucrose -.Sap-Stacks A-/6-& 1ST SAr\PLIN$> Sucrose-Tissue-Stalks 4 - J 6 -& -^3r5 5ampun^ — £wt> SA^PUNb M ‘DNi6H T FI6. Z\