THE ROLE OP TRANSPIRATION IN THE ABSORPTION AND
TRANSLOCATION OP MINERAL IONS IN PLANTS, AS
MEASURED WITH RADIOACTIVE CALCIUM AND
PHOSPHORUS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
RAFIQ AHMAD, B.Sc. (Alig), M.Sc. (Punjab)
The Ohio State University
1959
Approved hy
Adviser Department of Botany and Plant Pathology ACKNOWLEDGMENT
The ■writer wishes to express his gratitude to the faculty and graduate students of the Department of Botany and Plant Patho logy, The Ohio State University, for their great help during his
stay in the U. S. A. Special thanks are due to Dr. C. A. Swanson for his guidance and encouragement during this investigation and other graduate studies. The writer is also thankful to Dr. R. S.
Platt, Jr. for his help in preparing this manuscript and a critical reading of the draft. Thanks are also extended to Dr. C. G. Weis- haupt for critical reading of the manuscript.
The writer also wishes to express his appreciation to The
University of Karachi, Pakistan, for the grant of study leave, and to the Institute of International Education for the award of a travel grant to pursue these studies.
ii TABLE OP CONTENTS
Page
INTRODUCTION ...... 1
LITERATURE R E V I E W ...... 4
MATERIALS AND M E T H O D S ...... 8
EXPERIMENTS AND R E S U L T S ...... 14
Section 1 ...... 14 Experiments to investigate whether a low transpiration rate can he growth-limiting
Section 2 ...... 2 6 Experiments to determine the rate at which metabolically-absorbed phosphorus^ and c a l c i u m ^ become available for upward trans location into shoot
Section 3...... 41 Experiments to determine phosphorus^/ calcium45 ratio in various parts of plants as a function of time
DISCUSSION...... 48
SUMMARY ...... 59
LITERATURE CIT E D ...... 61
AUTOBIOGRAPHY...... 65
iii LIST OF TABLES
Table Page
1 Heights and dry weights of cotton plants sup plied with mineral ions during day only or night o n l y ...... 15
2 Dry weights of bean plants supplied with mineral ions during day only or night only (March 30 harvest l o t ) ...... 20
3 Dry weights of bean plants supplied with mineral ions during day only or night only (April 8 harvest l o t ) ...... 21
4 Heights and dry weights of bean plants supplied with mineral ions during day only or night only. 23 32 5 Leaching of P from the roots of cotton plants (experiment 4 A ) ...... 30 32 6 Leaching of P from the roots of bean plants (experiment 4B)...... 31 45 7 Leaching of calcium ^ from the roots of cotton plants (experiment 4 C ) ...... 32 45 8 Leaching of calciumfrom the roots of bean plants (experiment 4 C) ...... 33
9 Translocation of metabolically absorbed phos p h o r u s ^ from the roots of cotton plant as a function of time (experiment 5 A) ...... 38
10 Translocation of metabolically absorbed cal- cium45 from the roots of cotton plant as a function of time (experiment 5 B ) ...... 38
11 Translocation of metabolically absorbed phos p h o r u s ^ from the roots of bean plant as a function of time (experiment 5 C) ...... 39
12 Translocation of metabolically absorbed cal cium^ from the roots of bean plant as a function of time (experiment 5 B ) ...... 39
iv LIST OF TABLES (continued)
Tahle Page
A C "5 0 1 3 Distribution of calcium and phosphorus in various parts of hean plants ...... 43
1 4 Phosphorus^ and calcium^ content in different parts of hean plant expressed as a per cent of the total phosphorus32 ang calcium45 present in each plant at h a r v e s t ...... 54
1 5 Ratio of phosphorus"^ and calcium^ in different parts of the p l a n t ...... 58
v LIST OF FIGURES
Figure Page
1 Method of support for "bean seedlings growing in a shallow tank of culture s o l u t i o n ...... 10
2 Comparison of growth of cotton plants supplied with mineral ions during the day only or night only . . . * ...... 1 6
3 Arrangement of hean plants growing in the greenhouse...... 19
4 Bean plants showing no difference in heights if supplied with -g-X normal concentration of Hoag- land solution during the day only or night only. 22
5 Comparison of growth of hean plants supplied with mineral ions during the day only or night o n l y ...... 24
6 Apparatus designed to leach out non-metaholically absorbed compounds containing phosphorus^ ana c a l c i u m 4 5 from the "outer space" of the roots . 27 32 7 Leaching of phosphorus from excised roots of cotton and hean plants...... 34 A5 8 Leaching of calcium from excised roots of cotton and hean plants...... 35
9 Translocation of metaholically ahsorhed ions . . 40
10 View of hean plants growing in aerated culture solution in controlled environment room .... 42 45 11 Ratio between the activities of calcium and phosphorus^ (Ca/P) in different parts of plants 56
vi INTRODUCTION
The precise role of transpiration in the upward transloca tion of mineral salts in plants has been the subject of much dis cussion. It is known that inorganic ions absorbed by the roots are translocated upward predominantly in the xylem, although the specific channels in the xylem, that is, the cell types through which the bulk of this movement takes place, have never been delineated with certainty. However, it is reasonably probable that most of the ions are carried along by solvent drag forces through the walls and lumina of the tracheids, and, in such species as contain trachea, through these cells as well.
It follows from this view that an increase in transpiration rate, to the extent that it accelerates the movement of water up ward through these tracheary elements, should similarly increase the rate of transfer of ions from the roots to the shoots. There are, however, many complicating factors which can, and probably often do, obscure this simple relationship.
One possible factor is the re-export of ions from the leaves by way of the phloem toward the roots, followed by re-absorption into the xylem. In this case, increased transpiration rates may be ex pected to increase the rate of "cycling," but will not result in any appreciable net increase of ions in the shoot. Whether accelerated rates of cycling do result from increased transpiration rates has never been demonstrated experimentally, however. On the other hand, no evidence exists for excluding this possibility.
Another possible factor may be the "release rate" from the roots. Transfer of ions from the cortical cells of the roots and
other cells external to the xylem into the root and stem xylem may be a rate-limiting step. In this case, accelerating the velocity
of upward transport of water in the xylem would merely serve to re
duce the concentration of the ionic load in the "transpiration
stream." The quantity of ions transported to the shoot would be
independent, therefore, of the transpiration rate. That this pos
sible relationship obtains to a degree at least is suggested by the work of Hauber (1953)*
It is evident from these considerations that the rate of xylary transport of ions into the shoot as a function of trans piration rates involves complex variables, such as the physiological
status of the roots; the confused status of the problem in the lit
erature is therefore understandable. With the hope of clarifying a
few of these relationships, a study has been made of the role of transpiration in the upward movement of ions into the shoot. The
specific questions to which answers have been sought are as follows;
1. What is the long-term effect of transpiration differ
entials on the rate of ion accumulation in shoots? Can low rates
of transpiration be growth-limiting because of deficient mineral
ion supplies reaching the shoot?
2. How rapidly does the fraction of mineral ions absorbed
metabolically by the roots (the so-called "inner space" or "apparent
non—free space" fraction) become available for upward transport into the shoot? (The assumption is implied in this question that only
the ions in the "outer space"'*' or "apparent free-space" of the root
cells are carried along by solvent drag forces into the stem, and
hence ions absorbed into the "inner space" of the roots are at least
temporarily trapped. The question then becomes: How long are the
inner space ions trapped?)
3. Is the answer to question 2 significantly different for
anions vs. cations? (The negative charge on exchange sites in the
"outer space" of the root cells should materially affect the migra
tion velocity of cations as opposed to anions.)
4. If two isotopes ( P^ and Ca^) are supplied simultane
ously to the roots, how uniform is the ratio of these isotopes in
the different leaves on the plant after a short period of transpi ration, and how does this ratio vary with time? It is implicit in
this question that if mineral ions (represented here by radioactive phosphate and calcium ions) are transported to the leaves predomi nantly in the transpiration stream, the ratio of these ions should be constant for the different leaves unless there are complicating
selective exchange reactions in transit or redistribution of the mobile ions from the leaves.
^"Epstein defines "outer space" as "The space to which in organic ions have free and reversible access by diffusion." LITERATURE REVIEW
This field of study had heen extensively reviewed "by Curtis
(1935)» Kramer (l949> 1956), and Epstein (1956). Steward (1935 and
Gregory (1937)» while dealing with mineral nutrition of the plants, have emphasized the importance of metabolic activities of roots in the absorption of salts. By exposing corn and bean plants to dif ferent humidities to vary the rate of transpiration, Freeland (1937) and Wright (1939) demonstrated that an increase in the absorption of water resulted in an increase in mineral uptake. Butler (1953) car ried out a series of experiments on wheat to determine the influence of transpiration on the rate of retention of chloride and potassium by the root. Both ions were present in relatively greater concen tration in decapitated roots than in the roots of the intact plant.
Hylmo (1953) also has shown that degree of absorption of calcium is directly proportional to the amount of water transported in the transpiration stream.
Steward et_ al. (1936) gave more importance to the metabolic activities of the roots in salt absorption. They found a definite correlation between the aerobic respiration and accumulation of po tassium bromide in the roots as well as in stem tubers. Crafts and
Broyer (1938), working on squash, reported that the cortical cells of the absorbing region of the roots have a higher metabolic ac tivity than the living cells of the stele. This is said to favor diffusion of ions from the cortical region into the stele along an activity gradient. The subsequent movement of solute up into the shoot might he with the transpiration stream, hut they assert that the metabolic condition of the shoot is still the primary control ling factor. Steward et_al. (1942), in a study of accumulation and translocation of rubidium bromide in barley, mention that some kind of stimulus which keeps the root apices metabolically active is transmitted to them from the shoot. This causes supposedly absorp tion and translocation of salts. Experiments of Broyer and Hoag- land (1943) are important in this respect. They supplied mineral nutrients to three series of barley plants according to the follow ing plans (l) plants were supplied mineral nutrient during a 12-hour dark period each 24 hours; (2) a set of plants was supplied with mineral nutrients during the 12 hours of light each day; (3) a third set of plants was supplied with mineral nutrient continuously. At the end of three weeks the concentration of different salts in the roots and shoots was determined. They concluded that "On the whole, the amounts of ion absorbed were roughly the same whether the nu trients were available during the day or only during the night."
The concentration of salts in the plants getting nutrients continu ously was roughly double that of plants in the other series. These results suggest that most of the salts absorbed are translocated into the shoot, regardless of the rate of transpiration,
Hope and Stevens (1952) introduced the concept of "apparent free space" in roots. They defined it as "The region of the tissue to which electrolyte has access by diffusion." Epstein (1955) bas designated the same portion of a root as "outer space," in contrast 6
to "inner space" which he describes as the protoplasmic fraction of
the tissue capable of accumulating electrolytes metabolically, against
the a concentration gradient. There are various interpretations of
anatomical structure included in "outer" and "inner" space (Hope and
Stevens, 1952; Butler, 1953; Hylmo, 1953). Kramer (1956) considers
apparent free space primarily in terms of the amount of material which diffuses freely into and out of the tissue rather than in
terms of volume occupied. The volume of "outer space" has been de
termined in various plants by Hope and Stevens (1952), Hylmo (1953),
Butler (1953), and Epstein (1955, 1956) by titrimetric, potentiomet- ric, colorometric and radioactive tracer methods. Epstein (1944), with bromide-82 ions, was able to show that an enzymatic or enzyme like carrier mechanism exists in metabolic "fixation" of bromide in
the roots. Epstein and Leggett (1954) treated excised barley roots with labeled strontium which was later leached out in various cation
exchange solutions. In this way they distinguished two mechanisms
of salt absorption? (l) exchange absorption and (2) active trans port. The former is a simple diffusion while the latter involves
ion transport by means of a carrier or binding system (Jacobson
et al., 1950)• In another paper Epstein (l956) mentioned that radioactive sulphate absorbed by the active transport mechanism is mostly unavailable for translocation whereas all sulphate which gets up to the shoot, in the light, comes from the external solution by way of outer space.
In view of the above mentioned conflicting reports (Free
land, 1937; Wright, 1939; Butler, 1953; Hylmo, 1953, and others, as opposed to Steward et al., 1936; Crafts and Broyer, 1938; Broyer and Hoagland, 1943, and others), it was considered that further studies of the classic problem of transpiration in relation to salt absorption should be carried out. MATERIALS AMD METHODS
Cotton plants (Gossypium hirsutum L«) and Dean plants
(Phaseolus vulgaris L.. var. Black Valentine) were used in all ex periments reported in this investigation.
Acid delinted cotton seeds were obtained from Gro-Mor Cor poration, Hartwell, Georgia, and were separated in uniform-size lots. After soaking for one hour in distilled water, the seeds were placed in culture dishes on moist vermiculite for germination.
The culture dishes were placed in an incubator at 35°C which is within the optimum temperature range (33° to 36°C) recommended for the germination of cotton seed (Arndt, 1945)* The vermiculite was kept sufficiently moist, with particular concern for the high water requirement of the cotton seed during the germination (Eaton, 1955)*
The seedlings were kept in the incubator for about four days until they were long enough for transplanting into solution culture. At this stage the cotyledonary leaves were still enclosed within the seed coat. Mineral nutrient solution was prepared according to the formulation given by Meyer, Anderson, and Swanson (1955)* Micro nutrients were added according to the formulation of Arnon (1938), and iron was supplied in the form of ferric-potassium ethylenedia- mine tetraacetate (Jacobson, 1951)• The nutrient solution was changed once a week, and the solution level was kept constant by adding distilled water as required. Seedlings of uniform size were transferred, into one-quart Mason jars painted "black on the outside and -wrapped with aluminum foil. Each was fitted with a cork lid having two holes, one for receiving the aerator and the other for fixing the plant. The jars were placed in a controlled environ ment room and connected to a compressed air manifold. Sylvania
P. 96, TB/cw cool white fluorescent tubes, supplemented with Ken-
Rad 60-w tungsten bulbs, were used for illumination. The illumi nation intensity at the top of the plants was about 1000 P.O. A light period of 12 hours at 80°P was alternated with a 12-hour dark period at 70°F. The relative humidity of the room ranged between
30 -and 40 per cent during the course of present investigations. Cot ton plants having at least five fully expanded leaves (including two cotyledonary leaves) were used.
Bean seeds (Phaseolus vulgaris L., var. Black Valentine) were supplied by Capitol Seed and Garden Center, Columbus, Ohio.
The procedure with the beans was the same as that with cotton ex cept the experiment was carried out in the greenhouse instead of controlled environment room. The seeds had sprouted by the seventh day after sowing, and by the tenth day were tall enough for trans planting into culture solution. Seedlings of the uniform size were inserted through small corks held over a shallow tank of culture solution (Figure l). To avoid any metal contamination of the nu trient solution, the tank was painted with asphaltum paint. The culture solution was uniformly aerated by means of perforated Tygon tubes fixed in the bottom of the tank. The variations of photo period, temperature, light intensity, and relative humidity in the greenhouse during the course of e:rperimentation will be given in 10
wm
Figure 1.— Method of support for hean seedlings growing in a shallow tank of culture solution. 11 connection with the experimental results. Bean plants containing at least four fully expanded leaves (in addition to the two primary leaves) were used in most of the experiments.
Selection of Isotopes
Selection of phosphorus"^ and calcium^ as tracers in the absorption studies was the result of the following considerations.
The half-lives of phosphorus^ (14,3 days) and of calcium^ (152 days) are sufficiently long so that detectable radioactivity per- 32 sists during the experimental period. Phosphorus is produced by
op op S (n,p)P reaction in the uranium pile and is available in rela tively pure state. It decays by emission of a hard negative beta, having an upper energy limit of 1,701 Mev. Its assay presents no problem of self absorption unless the thickness of the sample is in excess of 50 mg/cm (Kamen, 1957), Samples can be easily counted by the end window type G-M counter. Phosphate is one of the most essential, highly mobile, naturally occurring anions in the plant tissue, therefore an undertaking of its translocation is of sig nificance in understanding the physiology of the plants. 45 Calcium is produced in the uranium pile reactor by
Ca^(n,y)Ca^ reaotion. The stock solution obtained from Oak Bidge was diluted so that one milliliter contained between 30 and 40 micro- curies for use in the present experiments. Calcium45 ^ decays by emis- tion of a soft negative beta having an upper energy limit of .260
Mev. This soft negative beta emission, however, creates a problem 45 / of self absorption. In the case of thick samples, calcium /mass ratio was used for the correction (Comar, 1955)* 12 45 Calcium is extensively used as a calcium tracer in "bio logical tissue because it is the only easily available radioactive isotope of calcium having the half life within reasonable range.
Calcium is also one of the most important naturally occurring cation in the plant tissue, notably as calcium pectate in the middle lamella of the cell wall.
Radioactive isotopes (phosphorus^, calcium^) used in this work were obtained from the Oak Ridge National Laboratories, and are listed in O.R.N.L. catalog as Item No. P-32-P-1 Processed High
Specific Activity and Item No. Ca-45"I>*'2 Processed High Specific
Activity, respectively.
Radioassay Procedure
Calcium^ and phosphorus"^ were applied to the plants as calcium chloride and phosphoric acid in the root solution. After a given period samples of root, stem, and leaves were harvested and prepared for assay by acid digestion. The tissue sample was placed in 50 ml beaker on a hot plate inside a hood. About 5 mL 6N nitric acidr was poured into the beaker. The beaker was then covered by a watch glass and gently heated. The acid fumes inside the beaker helped in digesting the part of the plant material not directly in contact with the liquid. The acid-digested solution of tissue was brought up to a standard volume and a 1-millimeter aliquot in a
1-inch stainless steel planchet was evaporated to dryness under an infrared heat lamp. In the leaching experiments, the Hoagland solu tion containing radioactive leachate was treated similarly. The amount of material on the planchets was so small that the correction 13 for self absorption was not needed. In other esqieriments where entire leaflets were acid-digested and dried directly on planchets, 45 the correction for self absorption in calcium counting was taken 45 into consideration. With samples containing a mixture of calcium 32 and phosphorus a differential absorption method for counting was adopted (Comar et al«, 195l)« Further details of this procedure will be given in connection with the experimental results.
Counting errors were evaluated statistically. As the stand ard deviation is stated to be equal to the square root of the number of counts (Comar, 1955)j an effort was made to have at least one thousand counts per sample if possible. In this way the standard deviation was reduced to about ^3 per cent. In those samples where counts were not greatly different from the background* the procedure was modified according to the recommendation of Comar (1955)• EXPERIMENTS AND RESULTS
Section 1
The following experiments were carried out to investigate the first question posed in the introduction; namely, whether a low transpiration rate can be growth-limiting because of.a reduced rate of supply of essential ions to the growing shoot tips.
Experiment No. 1
Cotton plants growing in one-quart Mason jars, under con trolled environment conditions, were matched together in pairs ac cording to size and number of leaves. One plant of every pair was kept in one-half the normal concentration of Hoagland solution dur ing the light period while the other was kept in distilled water.
During the dark period this treatment was reversed between the plants of each pair. In this way, one set of plants was in Hoagland solu tion during the light period and in distilled water during the dark period. The other set of plants was in distilled water during the light period and in nutrient solution during the night. The envi ronmental conditions and the photoperiod in the controlled environ ment room have been described earlier. The cotyledonary leaves of the plants kept in water during the light period started turning paler and began to fall earlier than the cotyledonary leaves of the plants kept in solution during the light period. The differentiation
14 15
in the rates of the growth "between the two sets of plants became
apparent after 20 days of the treatment* The heights of the plants were measured at the end of the experiment (Figure 2), and their dry weights determined. The complete data are as follows:
Cotton seed sown April 7» 1958 Transplanted in Mason jars April 12, 1958 (-g-X Hoagland solution) Treatment started April 241 1958 Plants harvested June 5, 1958 (Figure 2; Table l)
TABLE 1 HEIGHTS AND DRY WEIGHTS OF COTTON PLANTS SUPPLIED WITH MINERAL IONS DURING DAY ONLY OR NIGHT ONLY
Heights Dry Weights
Day, Water; Day, Solution; Day, Water; Day,Solution; Plant Night,Solution Night, Water Night,Solution Night, Water
1 20.0 cm 22.5 cm 2.09 gm 3.69 gm 2 17.5 31.5 1.86 7.60 3 20.5 37.4 3.03 11.22 4 20.0 32.4 2.79 9.52 5 25.0 30.5 3.57 5.91 6 21.0 30.0 2.35 4.34 7 21.0 25.5 3.12 4.23 8 22.0 24.0 2.79 3.75 9 21.4 29.0 1.93 4.37 10 19o5 32.0 2.02 7.93
Mean 20.75 cm 29.48 cm 2.54 gm 6.25 gm
Standard Error - .745 - 1.326 - .189 - .216
Experiment No. 2
Bean seeds were germinated in culture dishes on moist ver miculite and 4-day old seedlings were directly transplanted to one- gallon crocks containing distilled water cr culture solution. Each 16
Figure 2.— Comparison of growth of cotton plants supplied with mineral ions during the day only or night only. (Plant at extreme right was supplied with full concentration of Hoagland so lution continuously. The plants marked "S" were supplied with -JX normal concentration of Hoagland solution during the day only, and plants marked
"W" were supplied -gX normal concentration of Hoag land solution during the night only.) FIGURE 2. 18 crock was covered by a tygon-painted lid of masonite having one inlet for the aerator and three holes for securing three bean plants.
Eighteen crocks containing 54 plants were placed along side a brass air manifold which was then connected with the aerator tube of each crock (Figure 3). Half of the crocks were filled with distilled water and the others with half or quarter normal concentration of
Hoagland solution. Hay and night alternating treatment of the plants was the same as mentioned in the previous experiment. Transfer of plants from culture solution to distilled water and vice versa was carried out at sunrise and sunset. These experiments were performed in a greenhouse during the months of March and April, 1959* The en vironmental conditions of the greenhouse were as follows:
Sunrise (average) 5!30 A.M. (in April) :12 A.M. (i6:12 (in March) Sunset (average) 6:05 P.M. (in larch) 6:40 P.M. (in April) Light intensity about 10,000 F.C. (bright sunny days) (illumination) 4,000 F.C. (cloudy days) Temperature maximum temperature ranged between 90-100°F minimum temperature ranged between 60-70°F Relative humidity about 60$ at night and about 20$ on bright sunny days
Bean seeds sown March 6, 1959 Transplanted in crocks March 14, 1959 (•§X Hoagland solution) Treatment started March 18, 1959 First lot of plants harvested (Table 2) March 30, 1959 Second lot of plants harvested (Figure 45 Table 3) April 8, 1959 19
Figure 3.— Arrangement of bean plants growing in the greenhouse. Crocks containing distilled water alternate with the crocks con taining culture solution in each row. At the sunrise or sunset the tygon-painted masonite lids supporting the plants were transferred from the crocks containing culture solution to the crocks containing water, and vice versa. 20
TABLE 2 DRY WEIGHTS OP BEAN PLANTS SUPPLIED WITH MINERAL IONS DURING DAY ONLY OR NIGHT ONLY (MARCH 30 HARVEST LOT)
Plants Day Water, Night Solution Day Solution, Night Water
1 3.60 gm 4.3 gm
2 3.84 4.71
3 3.46 3.56
4 3.48 2.96
5 3.33 4o09
6 2.91 4.49
7 2.58 4.77
8 3.7 6 3.55
9 3.31 3.41
10 4.11 3.22
11 2.98 4.99
Mean 3.48 gm 4.36 gm
Standard Error - .106 - .202 21
TABLE 3 DRY V/EIGHTS OF BEAN FLAM'S SUPPLIED WITH MINERAL IONS DURING DAY ONLY OR NIGHT ONLY (APRIL 8 HARVEST LOT)
Plants Day water, Night Solution Day Solution, Night Water
1 7.71 gm 9.42 gm
2 6.65 8.92
3 7.44 6.44
4 6.96 8.52
5 5-59 8.77
6 4.64 8.27
7 6.80 6.98
8 7.58 4.15
9 6.53 6.26
10 8.39 8.33
11 5.41 10.16
12 6.72 7.93
13 5.27 7.74
14 5.49 8.17
15 5.21 7.07
Mean 6.42 gm 7.80 gm
Standard Error - .284 -3.80 22
Figure 4»— Bean plants showing no differ ence in heights if supplied with -§X normal con centration of Hoagland solution during the day only or night only. (Compare with results of similar experiments in -|*x normal concentration Hoagland solution given in Table 4 and Figure 5.) 23
Experiment No. 3
Bean seeds sown March 3 0 , 1959 Transplanted into crocks April 7» 1959 (•J-X Hoagland solution) Treatment started April 8, 1959 Plants harvested April 25, 1959 (Figure 5; Tahle 4 )
TABLE 4 HEIGHTS AID DRY WEIGHTS OF BEAR PLAITS SUPPLIED WITH MINERAL IONS DURING DAY ONLY OR NIGHT ONLY
Heights Dry Weights
Day Day Day Day Water, Solution, Water, Solution, Night Night Night Night Plants Solution Water Solution Water
1 28.9 cm 36.0 cm 2.36 gm 3.59 gm 2 24.0 50.5 - 1.88 3.42 3 34.6 37.8 1.83 4.62 4 26.4 41.5 1.97 3.57 5 31.5 39.0 1.71 3.00 6 33.0 60.5 2.10 4.20 7 26.8 52.5 2.00 3.85 8 26.0 40.2 1.70 3.31 9 29.5 44.0 2.11 3.35 10 29.0 48.0 2.01 3 063 11 31.0 36.5 1.91 3.55
Mean 29.15 cm 44.22 cm I .96 gm 3.64 gm
Standard Error - .888 - 2.420 - .189 1 .445
It is to he noted that in all the experiments reported ahove, the growth of plants supplied with mineral ions during the light period (high transpiration) was distinctly greater than that of plants supplied with mineral ions during the dark period (low transpiration). 24
Figure 5«— Comparison of growth, of hean plants supplied with mineral ions during the day only or night only. (Plants marked "S" were supplied with -J-X normal concentration
Hoagland solution during the day only, and the plants marked "W" were supplied with ■fX normal concentration Hoagland solution during the night only.) FIGURE 26
Section 2
The next problem was to determine the rate at which various
metabolically absorbed salt fractions became available for upward
\ 0 —— translocation into the shoot. Phosphate (as HP 0. ) and calcium d5++ (as Ca" ) were used as a representative anion and cation, re
spectively.
Experiment Ho. 4
Excised roots chosen for uniform size were used to determine the time required to leach out all the phosphorus"^ and calcium^
absorbed in the "outer space" of the roots. The experimental setup
is shown in Figure 6. A water bath was covered by a plastic plate
designed to support several test tubes immersed in the bath. Each
tube was fitted with a glass tube aerator. The excised roots were placed in test tubes containing either 50 ml of Hoagland solution minus phosphorus or 50 ml of Hoagland solution minus calcium. The
roots of one plant were placed in each tube. At zero time 30 to
40 microcuries of either phosphorus"^ or calcium^ were added to
each tube. The roots were kept for one hour in the radioactive
solution, after which they were rinsed thoroughly with distilled water and transferred into 40 ml of complete Hoagland solution.
Every hour the solution was replaced and the activity of the leachate
tested according to the procedure given earlier. This process was
continued until the activity in the leachate was negligible. Results
from replication of these experiments were in excellent agreement.
Tables 6-9 indicate the rate at which activity is leached from the
outer space. The results are also shown graphically in Figures 7 and 8. 27
Figure 6.— Apparatus designed to leach out non-metabolically absorbed compounds con taining phosphorus^ and calcium^ from the
"outer space" of the roots. 2 8
FIGURE 6. 29
These observations indicate that considerable amount of 32 phosphorus is leached out within six hours from the outer space of the roots. If this leaching is extended, only a very small 32 amount of phosphorus leaches out subsequently. In the case of 45 calcium it was observed that virtually all the amount of cal- 45 cium present in the "outer space" leached out within three hours.
Further investigations were based upon these results. It is as sumed for the purpose of later experiments in this section that only radioactive phosphate retained in the roots after six hours of leaching, or radioactive calcium after three hours, represents the "inner space" or metabolically absorbed fraction of these re spective ions. W CD £ w 4 H SB OJ ro H H- 0*3 O CD a CD ... to 0 0 6 2 4 0 0 3 9 3 4*. VO 00 Counts per Minute o o • • • H vn 4a 4* fo St. Deviation LEACHING OP P32 PROM THE ROOTS OP COTTON (EXPERIMENT PLANTS 4 A) vo 4a. 00 ON ON 8 VJ1 on • • • fo of Total Leachate
9 *9 9 VJl On vo
H
co II H —4 —4 Counts per Minute o O
o 006 O H • • • fo St. Deviation ro o vo VO ro H H i—1 H On Vn vn 00 fo of Total Leachate vo H ON H
4*. on 4a TABLE 5 H 00 M O O O Counts per Minute ►3 O o O H H r-» § • • • fo St. Deviation u> VJ1 OJ on Ph —3 “J —4 ON • • • • fo of Total Leachate 8 O H ON u> 4 CO
IO 4a ro Vn o CO Counts per Minute O o o o o o [O M H 4a • • • fo St. Deviation o on 00 4^ 4a on 4a- • • • • fo of Total Leachate Ov OJ ro OJ
ro ro H o on VO o o O Counts per Minute o o O ro ro ro vn • • • fo St. Deviation ro o UJ u > OJ ro • • • • fo of Total Leachate I-* 4* ro VO
ro H - 4 o on Counts per Minute o o o o o o ro ro o n • • • fo St. Deviation — 0 ro on r\) H ro ro • • • • fo of Total Leachate o ro ON ON oe W CD ICD •d H -P- vo ro H H p H* CRJ 0
t-o H H 6 TABLE H ro vo CO ro & vo Counts per Minute H vo -P* V o vn o vn o o o O o y? u> H* . • 0 • B ro ro ro VO fo St. Deviation CD vo co -p. 00 -0 vo 00 —4 W • • • « • fo of Total Leachate o vo ro vn ro § B m —j vo 00 -0 o -P> vn VO vo H vo vn Counts per Minute o O vn vn o OO O • # . • vo V o vo V o fo St. Deviation —4 ro •p» ov vn -P> -p* vn •• • • • fo of Total Leachate -P> H vo V o
1 —J OV 8 1 V O ro Counts per Minute OV 1 o V o vn I o O O o o •• vn -P* vo1 V O VO fo St. Deviation O vni ov vo -P- vo vn1 V O -p. . • • « fo of Total Leachate ro -a H1 —1 vn
vn OV ov vn vn ro vo ov CO H vn ro Counts per Minute vn o OO O o O o ov # • • • -P> -P» vo -p> fo St. Deviation ro o ro vo vo VO •P- • -p>•• • fo of Total Leachate vo -p* ov vn H T£ W ro
H ro OJ UJ v n O Counts per Minute ro o O 8 o o o o H Q • ». CD ov v n f St. Deviation O vn ro —J *1 o £ o CD vo CD 03 CD H ON ON a • • • . f of Total Leachate OJ •—0 00 o v vn
ro o j s oo ro t-1 Counts per Minute V n — 3 O o o o W H3
oj ro h I I • • • f St. Deviation ro U i td .£>• H —3 O -J r-3 H- o ©g 03 ■—0 —3 Co • • • fo of Total Leachate re H 03 CO -J W § H§ m H M u> o \ vn Counts per Minute vn O -o CO O O vn
vn ro ro ... fo St. Deviation i_u vn vn
-t=> ro vn -fc» o • • • f of Total Leachate O OJ vn 4^
tej tel teS CD ro CD CD m OS os, OS M H H 1 M Counts per Minute H- H* H- M- 0>3 OS 0*5 OS H- H- H- H- o' o' o' o' t-“ H H M CD CD CD CD ZZ W CD w H H fo o j ro Cft O o P> cf CD CD
ro M H H VJl
ro O o Counts per Minute LEACHING OP CALCIUM45 PROM THE OP ROOTS (EXPERIMENT BEAN PLANTS C) 4 o U 1 o o o o
• • o . — 3 CO 00 fo St. Deviation H
00 C o 00 00 ro 1— 1 -p» H • ••• fo of Total Leachate VJl CO VJl OJ
OJ H ro ro •—J OJ Counts per Minute O o o o o o TABLE 8
H ro ro ••• ro ►3 -0 o fo St. Deviation H- ro CO CDi
m H ro H 8 H • vo OJ fo of Total Leachate 4 • OJ • • 03 - 0 o n OJ
H VJ1 O VO Counts per Minute O VJl O o O O
\J1 w w • • • fo St. Deviation OJ - J O O O J CO
On VJl VJl U 1 e • • fo of Total Leachate voi -o oo ro
tej 04 tej tej CD CD CD CD Cft (ft Cft (ft H HMH Counts per Minute M- H*H-H- Cft (ft (ft (ft H- H*H*H- O' o ’ o' o' H M H t-J CD CD CD CD ££ PERCENT OF TOTAL ACTIVITY LEACHED 60 50 40 20 30 70 0 tG E 7. Ft GU RE ECIG F HSHRS RM EXCISED PHOSPHORUS FROM OF LEACHING O T O CTO AD EN PLANTS BEAN AND COTTON OFROOTS I 2 TIME 34 3 (HOURS) 2 3 4 5 COT TO BEAN
6 PERCENT OF TOTAL ACTIVITY LEACHED 40 30 50 60 90 70 FIGURE 8. ECIG F ACU FO ECSD ROOTS EXCISED FROM CALCIUM OF LEACHING F OTN N BA PLANTS BEAN AND OF COTTON TIME (HOURS) 5 3 45 2 COTTON BEAN
36
Experiment Ho. 5
Plants of uniform size were transferred to 100 ml wide- mouth bottles. These "bottles were connected with aerators and placed inside a nine cuMc foot humidity chamber made from enam eled Masonite. The mist created by four nebulizers in the chamber was sufficient to maintain 100 per cent humidity. In the course of an hour, practically no transpiration was noted from a test cotton plant placed in a potometer and kept in this high humidity chamber.
The detailed outline of protocol for experiment No* 5 was as follows:
Period 1. Plants transferred to the high humidity chamber
(dark) for 1 hour in complete Hoagland solution, to permit a re duction in hydrostatic tensions within the plant and to reduce the transpiration rate to zero or near zero.
Period 2. Complete Hoagland solution replaced by Hoagland 45 32 solution containing 30 to 40 microcuries of calcium or phosphorus per 60 ml of solution (this solution otherwise free of calcium and phosphate ions). Plants were kept in this "hot" salt solution for one hour.
Period 3. At the end of period 2 the roots thoroughly rinsed, and the "hot" salt solution replaced by complete Hoagland solution. Hoagland solution replaced fresh every hour. Leaching 32 process carried out for six hours in the case of phosphorus and 45 for three hours in the case of calcium . End of this leaching period referred to as zero time. One plant harvested immediately.
Period 4. Remaining plants transferred to a light room under conditions favoring high transpiration rate. These plants harvested after one, four, and eight hours from zero time.
The sample plants were divided into roots and shoots which were assayed separately. The results are presented in the tables
9 to 12. A graphic illustration of these results is shown in
Figure 9» TABLE 9 TRANSLOCATION OP METABOLICALLY ABSORBED PHOSPHORUS32 PROM THE ROOTS OP COTTON PLANT AS A FUNCTION OP TIME (EXPERIMENT 5 A)
Duration of Period Root Shoot of High Transpirat i on Counts per i s t. io of Total Counts per i s t. io of Total Root/Shoot (Hours) Minute Deviation Activity- Minute Deviation Activity- Ratio
0 2156 2.1 74. 6 734 3.6 25.4 2.93 1 899 3.3 68.4 446 4.7 31.6 2.16 4 263 6.1 56.3 204 6.9 43.7 1.28 8 237 6.4 55.1 193 7.1 44.9 1.22
oj CO
TABLE 10 _ TRANSLOCATION' OP METABOLICALLY ABSORBED CALCIUM4-3 PROM THE ROOTS OP COTTON PLANT AS A FUNCTION OP TIME (EXPERIMENT 5 B)
Duration of Period Root Shoot of High Transpiration Counts per i s t. i of Total Counts per i s t. io of Total Root/Shoot (Hours) Minute Deviation Activity- Minute Deviation Activity Ratio
0 8400 1.9 73.6 3000 1.8 26.4 2.78 1 7700 1.1 62.6 4600 1.4 37.4 1.67 4 5300 1.3 51.4 5000 1.4 48.6 1.06 8 6300 1.2 52.5 5700 1.3 47.5 1.11 TABLE 11 TRANSLOCATION OP METABOLICALLY ABSORBED PHOSPHORUS32 PROM THE ROOTS OP BEAR PLANT AS A JUNCTION OP TIME (EXPERIMENT 5 C)
Duration of Period Root Shoot of High Transpiration Counts per i s t. io of Total Counts per i s t. i of Total Root/Shoot (Hours) Minute Deviation Activity- Minute Deviation Activity Ratio
0 128650 0.20 92. 6 10200 0.98 4.7 19.70 1 134050 0.26 88.7 17010 0.76 11.3 7.84 4 60805 0.60 53.1 53500 0.43 46.9 1.13 8 21750 0.67 14.0 132800 0.27 86.0 0.16 u> vo
TABLE 12 TRANSLOCATION OP METABOLICALLY ABSORBED CALCIUM45 PROM THE ROOTS OP BEAN PLANT AS A FUNCTION OP TIME (EXPERIMENT 5 D)
Duration of Period Root Shoot
Transpiration Counts per i s t. io of Total Counts per i St. i of Total Root/Shoot (Hours) Minute Deviation Activity Minute Deviation Activity Ratio
0 1738 2.3 95.8 95 9.0 4.2 22.80 1 1825 2.3 87.9 250 6.3 12.1 7.26 4 1414 2.6 60.4 925 3.2 39.6 1.52 8 631 3.9 43.4 825 3.4 56.6 0.76 PERCENT TOTAL ACTIVITY PERCENT TOTAL ACTIVITY 0, OR s-OU 4hHU SthHOUR HOUR 4th UR |st-HO HOUR , 0 0 IUE 9. FIGURE RNLCTO O MTBLCLY BOBD IONS ABSORBED METABOLICALLY OF TRANSLOCATION PHOSPHOR US PHOSPHOR 40 QHU |sf OR t OR RthHOU0 HOUR 4th HOUR f s | Q.HOUR ] SthHOUR 4thHOUR R U O fH /s HOUR o, O. OR tHU 4hHU SthHOUR 4th HOUR 5t;H0UR | HOUR 41
Section 3
To answer the final question the following experiments were
carried out to determine the phosphorus^/calcium^ ratio in the
different leaves, after various periods of time when the roots had
heen supplied with these isotopes simultaneously.
Experiment No. 6
Bean plants of uniform size were transferred from the tank
of culture solution into one-quart Mason jars each containing Hoag
land solution minus calcium and minus phosphorus. Complete Hoag
land solution containing stable isotopes was replaced by complete 45 Hoagland solution containing 40 microcuries of calcium and 20 32 microcuries of phosphorus per liter. This proportion for these
radioactive ions corresponds to the ratio recommended by Comar,
1955> experiment involving double isotope computations. Plants were kept in the control room under continuous light (Figure 10),
where the conditions were favorable for transpiration throughout
the entire time course of the eaperiment (60 hours). The time
schedule for plant harvest is shown in Table 13. Root, stem, and * individual leaves of every plant were assayed separately. For as
saying roots, stem, and first trifoliate leaf, the acid-digested
solution was brought up to 50 or 25 ml in volume and a 1-milliliter
aliquot was dried on a planchet. In this way the thickness of the
sample was kept to a minimum and the activity was reduced to a
range that could be easily counted. The total volume of acid-
digested solution of the other leaves was dried on the planchets,
and the correction for the self-absorption made according to Walter 42
Figure 10.— View of "bean plants growing in aerated culture solution in controlled en vironment room. (Bean plants growing in the radioactive culture solution were exposed to this light continuously during the term of the experiment.) 43
TABLE 13 DISTRIBUTION OP CALCIUM45 AND PHOSPHORUS32 IN VARIOUS PARTS OP BEAN PLANT
One Hour After the Application of Activity
32 45 Phosphorus Calcium ^
Thickness of Sample mgo per Corrected Plant Parts c.p.m,• C «jp •IQi. sq. cm. c #p *m.
Root 23250 20100 Negligible 20100 Stem 1200 Nil - — 1st Primary Leaf 47 Nil - - 2nd Primary Leaf 43 Nil - - 1st Trifoliate Leaf 525 Nil -- 2nd Trifoliate Leaf 132 Nil -- 3rd Trifoliate Leaf 28 Nil - - Apex Nil Nil —
Two Hours After the Application of Activity
Root 30900 19450 Negligible 19450 11 Stem 975 650 650 1st Primary Leaf 124 Nil - - 2nd Primary Leaf 108 Nil - - 1st Trifoliate Leaf 787 Nil -- 2nd Trifoliate Leaf 375 Nil - - 3rd Trifoliate Leaf 91 Nil - - Apex 15 Nil
Pour Hours After the Application of Activity
Root 32 625 15575 Negligible 15575 Stem 1425 1175 11 1175 1st Primary Leaf 195 25 10 43 2nd Primary Leaf 259 39 13.1 69 1st Trifoliate Leaf 1050 198 Negligible 198 2nd Trifoliate Leaf 663 187 22.2 935 3rd Trifoliate Leaf 72 Nil -- Apex 24 Nil - - 44
TABLE 13— (continued)
Eight Hours After the Application of Activity
32 Phosphorus Calcium^
Thickness of Sample mg. per Corrected Plant Parts c.p.m. c.p.m. sq., cm. c.p.m.
Root 25200 13850 Negligible 13850 Stem 1650 1650 11 1650 1st Primary Leaf 372 65 6.0 87 2nd Primary Leaf 408 99 6.1 133 1st Trifoliate Leaf 1575 425 Negligible 425 2nd Trifoliate Leaf 313 122 14.2 254 3rd Trifoliate Leaf 55 Nil -- Apex 30 Nil --
Twelve Hours After the Application of Activity
Soot 70875 5290 Negligible 5290 Stem 4050 4850 n 4850 1st Primary Leaf 859 329 13.1 685 2nd Primary Leaf 807 440 12.9 1551 1st Trifoliate Leaf 3037 2313 Negligible 2313 2nd Trifoliate Leaf 3517 1100 31.9 4400 3rd Trifoliate Leaf 1224 538 17.2 1280 Apex 159 140 Negligible 140
Eighteen Hours After the Application of Aotivity
Root 8175 10275 Negligible 10275 Stem 10747 130803 11 130803 1st Primary Leaf 1539 557 14.5 1237 2nd Primary Leaf 1816 863 13.2 1797 1st Trifoliate Leaf 7987 10438 Negligible 10438 2nd Trifoliate Leaf 4026 4442 32.2 17768 3rd Trifoliate Leaf 4542 2008 14.6 4365 Apex 756 61 Negligible 61 TABLE 13— (continued)
Twenty-four Hours After the Application of Activity
Phosphorus^ Calcium^
Thickness of Sample mg. per Corrected Plant Parts c.p.m. c.p.m. sq. cm. c.p.m.
Root 97050 38700 Negligible 38700 Stem 130803 14550 II 14550 1st Primary Leaf 2260 1490 13.1 2980 2nd Primary Leaf 1984 1318 12.9 2636 1st Trifoliate Leaf 6637 11788 Negligible 11788 2nd Trifoliate Leaf 6030 3085 22.5 9640 3rd Trifoliate Leaf 3492 2498 16.5 5677 Apex 405 132 Negligible 132
Thirty Hours After the Application of Activity
Root 5400 1895 Negligible 1895 Stem 8250 10750 11 10750 1st Primary Leaf 885 367 10.2 633 2nd Primary Leaf 879 502 12.0 965 1st Trifoliate Leaf 3112 2988 Negligible 2988 2nd Trifoliate Leaf 1929 1200 38.7 4800 3rd Trifoliate Leaf 486 445 15.7 988 Apex 106 56 Negligible 56
Thirty-six Hours After the Application of Activity
Root 17025 19325 Negligible 19325 Stem 8850 12150 II 12150 1st Primary Leaf 1767 617 16.5 1469 2nd Primary Leaf 5386 2078 22o9 6111 1st Trifoliate Leaf 3712 2888 Negligible 2888 2nd Trifoliate Leaf 2535 914 34o5 3046 3rd Trifoliate Leaf 732 47 6 9.1 767 Apex 195 102 Negligible 102 46
TABLE 13— (continued)
Forty-four Hours After the Application of Activity
Phosphorus^ Calcium^
Thickness of Sample mg. per Corrected Plant Parts c.p.m. c.p.m. sq. cm. c.p.m.
Root 58800 2350 Negligible 2350 Stem 114375 39225 If 39225 1st Primary Leaf 4077 2252 17.8 5361 2nd Primary Leaf 7194 3667 17.2 8730 1st Trifoliate Leaf 9600 21725 Negligible 21725 2nd Trifoliate Leaf 7471 3070 16.5 7309 3rd Trifoliate Leaf 5917 2553 6.7 3545 Apex 715 667 Negligible 667
Fifty-two Hours After the Application of Activity
Root 84150 32150 Negligible 32150 Stem 13650 18400 11 18400 ' 1st Primary Leaf 3238 2209 14.2 3545 2nd Primary Leaf 2547 1606 11.6 2920 1st Trifoliate Leaf 11661 13689 Negligible 13689 2nd Trifoliate Leaf 10029 3829 11.9 6961 3rd Trifoliate Leaf 7866 5560 30.4 22240 Apex 2079 732 Negligible 732
Sixty Hours After the Application of Activity
Root ;2 2 0 2 0 0 69950 Negligible 69950 Stem 2895 54705 11 54705 1st Primary Leaf 6729 3455 18.1 8426 2nd Primary Leaf 1590 696 15.1 1581 1st Trifoliate Leaf 22800 27350 Negligible 27350 2nd Trifoliate Leaf 47212 51763 11 51763 3rd Trifoliate Leaf 20619 5559 42.0 29257 Apex 8292 2927 3o6 3181 47
(1951) whenever necessary. The method of differential absorption
(Comar, 1951)i was used for the counting. An aluminum absorber of 2 about 55 mg/cm is reported to stop essentially all the beta of 45 32 energy of calcium , but reduces phosphorus activity to two-thirds of its total. The factor was determined empirically with an alumi- 32 num absorber and a pure phosphorus source. Accordingly, calcula tions for this study were made using the following equation:
2 C = T - P
*5 0 Where, P = Activity (counts per minute) due to phosphorus only.
A = Activity (counts per minute) of the mixed source as 2 measured after placing an aluminum absorber of 55 mg/cm
in thickness.
C si Activity (counts per minute) due to calcium^ only.
T = Total activity (counts per minute) of the mixed source
as measured without absorber.
The complete data are presented in the preceding tables, and a discussion of the probable significance of these results is given in the next section. DISCUSSION
The results of the experiments 1 to 3 show that plants kept in culture solution during the day and in distilled water during the night exhibit better growth in comparison with the plants treated reciprocally. Data given in Table 1 reveal that the difference be tween heights and dry weights of the two sets of plants are sig nificant to the .1 per cent level.
Prior to selecting the concentration of Hoagland solution used in experiment No. 1, a series of cotton plants were grown in complete, half, quarter, and one-eighth concentration of the macro- metabolic elements. Analysis of the growth of these plants showed that those growing in the half concentration grew only slightly less well than the plants growing in full concentration, but much better than the plants growing in one-fourth or one-eighth concentration.
In other words, the full concentration of Hoagland solutions is not much in excess of the concentration optimum for plant development.
Even though the full concentration of Hoagland solution was at least sufficient to supply the amount of salts necessary for healthy growth, there is also the possibility of a slight excess and perhaps some accumulation of the salts in the roots within the nontoxic limit.
If, as in the experiment of Broyer and Hoagland (1943), a series of plants are kept in full concentration of culture solution during the night and in distilled water during the day, it is likely
48 49
that an amount of minerals will be accumulated metabolically by-
active transport in "inner space" sufficient for optimal growth of
the shoot even during the period in distilled water. Root "outer
space" of course contains approximately the same concentration of
mineral ions as the external solution. Thus at the end of the nu
trient solution period the roots of the plants will probably be
saturated, and this may constitute an excess of ion with regard to
the mineral requirement of the shoot at that time. Subsequently,
when the plants are placed in distilled water, the metabolically
absorbed fraction of ions as well as the ions present in "outer
space" become available for translocation to the shoot, and shoots
are supplied with minerals irrespective of the mineral present in
the external solution. It appears reasonable, therefore, that
Broyer and Hoagland (1943) might not have found any difference in
growth between the two series of plants, treated reciprocally.
This study has shown that metabolically absorbed ions are
readily translocated to the shoot under conditions of high trans
piration rates. Furthermore, experiments No. 1 to 3 indicate that
accumulation probably does not occur in plants kept in a slightly
suboptimal condition of culture solution. The half concentration
culture solution used in experiment No. 2 was not quite growth-
limiting and there was accordingly only a relatively small differ
ence in growth. Some plants of both series were harvested while
still in the vegetative condition (Table 2), the others after flower
ing and fruiting. In neither series was there much difference in
heights between the plants undergoing different treatments (Figure 4)* 50
Differences in dry weight between two series of plants, however, presented in Tables 2 and 3 are significant to 0.1 per cent levelo
Bean plants, in experiment No. 3, were kept in one-fourth
concentration Hoagland solution, clearly suboptimal with respect to the macronutrient concentration required for rapid growth. These plants began to show a differential growth rate within a week from transplanting. The difference between the height and dry weights
of these plants (Figure 5), shown in Table 4> is significant to less than 0.1 per cent level.
A comparison between day and night rates of transpiration
of bean plants in the greenhouse showed that the rate of transpira tion during the day was about ten times that at night under the conditions prevailing at the time this experiment was conducted.
The results of the experiments described in section one clearly in dicate that the availability of ions in the shoot, expressed in terms of shoot length and dry weight, is greater in the plants kept in culture solution during the day. This strongly suggests that high rates of transpiration promote the translocation of ions. Low rates of transpiration, occurring during only the period of time that mineral salts are available, has a growth-limiting effect. It may be concluded that transpiration facilitates translocation of mineral
ions. The studies reported by Freeland (1937)> Wright (1939)>
Butler (1953), and Hylmo (1953) are in agreement with these results.
The experiments described in section two constitute a measure ment of the rate at which metabolically absorbed fractions of cation and anion become available for upward translocation into the shoot. 51
■52 mm mm Phosphate (as HP 0^ ) and calcium (as Ca ^ ) were used as rep resentative anion and cation, respectively. Experiment Ho. 4 was conducted to determine the time required to leach out most of the phosphorus"^2 and calcium^ absorbed in "outer space." Similar leach ing of ions from the "outer space" has been a common technique for investigating the volume of "outer space" (Hope and Stevens, 1952;
Butler, 1953; Hylmo, 1953; Epstein, 1955> 1956). Tables 5 to 8 present data on the rate of leaching of phosphorus"^2 and calcium^ labeled material from the roots of cotton and bean plants. These results are further illustrated in Figures 7 and 8. It is evident that more than 50 per cent of the total activity was leached out within an hour, both with phosphorus"^2 and calcium^. The anion - \ o _ HP 0^ is leached more slowly over a longer period of time than the cation si45 in both cotton and bean plants. Ho ready explana tion for this difference is possible at present.
The protocol in the experiment Ho. 5 is based on the results of experiment Ho. 4» The schedule is such that nearly all the cal cium^ and phosphorus^2 activity is leached out from the "outer space" during the period of minimum transpiration. After this leaching is complete the plants are exposed to conditions of high transpiration. Results presented in the Tables 9 to 12 and Figure 45 9 show that the metabolically absorbed fraction of calcium ^ and 32 phosphorus is immediately available for translocation to the shoot under conditions of high transpiration. Presumably a portion of metabolically-absorbed ions are released into "outer space" either as ions or in bound state, and might then be carried along by sol vent drag forces in the water moving through the "outer space" to 52
the shoot. However, the plants harvested at zero hour show that
a very small amount of calcium^ and phosphorus^ is translocated
into the shoot during the second and third periods of the protocol
given on page • Whether this resulted from a slow transpiration
rate (check plants measured potometrically showed zero to negative
transpiration rates, however, in high humidity chamher), or from
slow cycling of water between shoot and root, or from other causes,
cannot be stated. When, in the fourth period of protocol, the plants were exposed to high transpiration condition, the metabolically trap ped ions become available for translocation without delay (Tables 9 to 12). No significant difference between the rate of translocation
of metabolically absorbed cation vs. anion was noticed in individual plants, but the rate of translocation of ions in cotton was found
quite different from that in bean. It can be concluded that the rate of translocation of metabolically absorbed ions depends upon
the root metabolism as well as the rate of the transpiration stream
of the individual species. These results are contrary to more or less similar experiments reported by Epstein (1956). He did not find any translocation of metabolically absorbed ions from "root
inner space" to the shoot. op Last experiment (Ho. 8)was designed to determine phosphorus / 45 calcium ratio in different parts of the plant after exposing the 3 2 _____ 45++ roots to a simultaneous supply of HP 0^ and Ca . The results
are presented in Table 13. The later table presents the amount of
calcium^ and phosphorus^ present in each part as a per cent of
total amount of calcium^ or phosphorus^ present in the plant at
that time. These data from plants harvested one hour after isotope 53 45 application to the roots show that calcium ^ is not translocated into the shoot at all, although there appears to he considerable amount of this isotope absorbed into the root. On the other hand, 32 7*9 per cent of the phosphorus activity is translocated into the shoot as a whole within the first hour.
In the plants harvested after the second hour only 3*3 per 45 cent of the calcium has been translocated to the stem, whereas 32 phosphorus in the shoot by this time is more than twice this 45 amount. The delay in translocation of calcium ^ to the shoot is presumably due to a delay in transport of this ion from the cortical region of the root to the stele or may result from the fact that a clearer delineation can be made between the "outer space" and "inner 45 space" calcium ^ fractions<, The delay might well be the result of an adsorption or exchange reaction in "outer space," thus account ing for the observed cation-anion differential. A comparison be tween the amounts of phosphorus^ and calcium^ activities in the 45 stem shows that translocation of calcium from stem to the leaves 32 is slower than that of phosphorus , and hence a greater percent- 45 age of calcium is retained in the stem. This retention of cal- 45 c ium J in the stem may also be attributed to selective exchange re actions.
Table 14 shows that detectable amounts of phosphate ac cumulate in the leaves two hours earlier than of calcium, where it undoubtedly enters into metabolic pool, particularly as sugar phos phate, and hence is rapidly redistributed. This would cause the ratio phosphorus^/calcium^ in various parts of the plant to be TABLE 14 PHOSPHORUS32 AID CALCIUM45 CONTENT IN DIFFERENT PARTS OP BEAN PLANT EXPRESSED AS A PER CENT OP THE TOTAL PHOSPHORUS32 AND CALCIUM45 PRESENT IN EACH PLANT AT HARVEST
Time of 1st Pri- 2nd Pri- 1st Trifo- 2nd Trifo- 3rd Trifo- Harvesting Root Stem mary Leaf mary Leaf liate Leaf liate Leaf liate Leaf Apex After Application Per Cent Per Cent Per Cent Per Cent Per Cent Per Cent Per Cent Per Cent of Activity ,32 45 ,32 (Hours) Ca Ca45 P32 Ca45 P32 Ca45 P32 Ca45 P32 Ca45 P32 Ca45 P32 Ca45
1 92.1 1 0 0 , 4.7 - .02 - .01 - 2.0 - .5 - .67 - - - 2 92.5 96.7 2.9 3.3 .37 - .32 - 2.3 - 1.1 - .27 - .24 - 4 89.8 86.5 3.9 6.5 .53 .23 .71 .38 2.8 1.1 1.8 5.1 .19 - .27 - 8 85.1 84.4 5.5 10.0 1.2 .53 1.3 .81 5.3 2.5 1.0 1.5 .18 - .42 - 12 83.8 65.7 4.7 10.9 1.0 1.5 .9 3.4 3.5 5.1 4.1 9.8 1.4 2.8 1.4 .8 18 78.8 42.5 5.9 12.8 1.1 1.5 1.3 2.2 5.8 13.0 2.9 22.2 3.3 5.4 .9 2.5 24 74.8 44.9 9.1 16.8 1.7 3.4 1.5 3.0 5.1 13.6 4.6 11.1 2.6 6.5 .6 .7 30 77.5 47.2 11.8 26.7 1.2 1.5 1.1 1.4 4.4 7.4 2.7 11.9 .7 2.4 .6 1.5 36 42.3 42.1 22.0 26.4 4.3 3.2 13.3 13.3 9.2 6.2 6.3 6.6 1.8 1.6 .8 .6 44 28.2 21.3 54.9 35.5 1.9 4.8 3.4 7.9 4.6 19.7 3.5 6.6 2.8 3.2 .7 1.0 52 62.2 31 o9 10.0 18.2 2.3 3.5 1.8 2.9 8.6 13.6 7.4 6.9 5.8 22.0 1.9 1.0 60 61.7 31.7 8.1 13.0 1.8 3.8 .4 .7 6.3 12.4 13.2 23.5 5.7 13.2 2.8 1.7 different than in transpiration stream (Figure ll)• The various ratios of phosphorus^/calcium^ in different parts of the plant are presented in Table 15* The proportions are definitely not con stant, nor do they vary in any obvious simple way.
It would appear, as suggested earlier, that the translocation of phosphate and calcium are not simple instances of mass flow in the transpiration stream, but instead involve a complex of processes, including mass flow as an important central mechanism, but modified by selective adsorption, selective ion exchange reactions, and pro bably other different specific rates of redistribution. 56
FIGURE II 45 RATIO BETWEEN THE ACTIVITIES OF CALCIUM a PHOSPHORUS^ Ca/P) IN DIFFERENT PARTS OF PLANTS
0.66 0. 82
0.22 0.2 6
0.86 0.3 0.47
ONE HOUR TWO HOURS FOUR HOURS
0.88 0.0 8 0.31 0.2 3 0.32 0.7 91.92 0.8 0. 9 8 0.54 0.24 1.25 EIGHT HOURS TWELVE HOURS EIGHTEEN HOURS 57 (continued) 45 RATIO BETWEEN THE ACTIVITIES OF CALCIUM a 32 PHOSPHORUS ( Co/P) IN DIFFERENT PARTS OF PLANTS 0.32 W 0.28 0.52 rt' I. 22 1.37 1.32 I. 090.71 0. S3 1.13 0.39 0.35 TWENTY-FOUR HOURS THIRTY HOURS THIRTY-SIX HOURS 0 .9 3 0.3 5 o.3 e d 1 to 0.42 1.34 0. 98 1.31 1.21 1.0 9 I. 88 0.99 0.39 0.3 8 0.31 FORTY-FOUR HOURS FIFTY-TWO HOURS SIXTY HOURS TABLE 15 RATIO OP PHOSPHORUS32 ABB CALCIUM4^ ijj BIFFERENT PARTS OP THE PLANT Time of Har vesting After Application of Activity 1st Primary 2nd Primary 1st Trifo- 2nd Trifo- 3rd Trifo- (Hours) Root Stem Leaf Leaf liate Leaf liate Leaf liate Leaf Apex 1 1 0.86 1 0.00 1 0.00 1 0.00 1 0.00 1 0.00 1 0.00 0 0.00 2 1 0.30 1 0.66 1 0.00 1 0.00 1 0.00 1 0.00 1 0.00 1 0.00 4 1 0.4T 1 0.82 1 0.22 1 0.26 1 0.18 1 1.41 1 0.00 1 0.00 8 1 0.54 1 1.00 1 0.23 1 0.32 1 0.2 6 1 0.81 l 0.00 1 0.00 12 1 0.24 1 1.19 1 0.79 1 1.92 1 0.76 1 1.25 1 1.04 1 0.88 18 1 1.25 1 0.31 1 0.80 1 0.98 1 1.30 1 4.41 1 0.96 1 0.08 24 1 0.39 1 1.22 1 1.31 1 1.32 1 1.77 1 1.59 1 1.62 1 0.32 30 1 0.35 1 1.30 1 0.71 1 1.09 1 0.96 1 2.48 1 2.03 1 0.28 36 1 1.13 1 1.37 1 0.83 1 1.13 1 0.77 1 1.20 1 1.04 1 0.52 44 l*Oo39 1 0.42 1 1.31 1 1.21 1 2.20 1 0.97 1 0.59 1 0.93 52 1 so.38 1 1.34 1 1.09 1 1.88 1 1.17 1 O .69 1 2.82 1 0.35 60 1 :0.31 1 0.98 1 1.25 1 0.99 1 1.19 1 I .09 1 1.41 1 0.38 SUMMARY A series of experiments were performed to study the effect of transpiration on the absorption and translocation of mineral ions in cotton and bean plants. During the light period one set of plants was kept in a mineral nutrient solution and another set in distilled water. During the dark, period the treatment of the two sets was re versed. For these experiments the mineral nutrient solutions were adjusted to a concentration in which the plants grow slightly less well than plants growing on full concentration. This presumably avoids ion accumulation in the "outer" and "inner spaces" of the roots to the extent that they would be available for translocation during the period when the plants are kept in distilled water. Plants kept in mineral nutrient solution during the period of rapid transpiration (day) grew much better than the plants kept in mineral solution during the night period of low transpiration. Results of these experiments strongly suggest that a higher rate of transpiration during the period of mineral salt availability is responsible for greater rate of translocation. Conversely, a low rate of transpiration during the period of mineral salt availability may be growth-limiting. Op In other experiments, phosphate (as HP 0^ ) and calcium (as C a ^ ++) were used to determine the rate at which metabolically absorbed ions in the roots become available for upward translocation. 59 60 Experiments were carried out in such, a way that almost all phos phorus^ and calcium^ from the "outer space" of the roots could he leached out under conditions of minimal transpiration. The plants were subsequently exposed to an environment favoring a high rate of transpiration. Hoots and shoots were separately radioassayed after various time intervals. Contrary to some previous reports it was found that metabolically absorbed ions become immediately available for upward translocation. The final series of experiments were designed to study the distribution of phosphate (as HP^20^ ) and calcium (as C a ^ ++) in various parts of the plant. The stable isotopes were replaced by these radioactive isotopes in the culture solution throughout the course of experiments (60 hours). The plants were kept in a con trolled environment room with continuous light under conditions that favored rapid transpiration. The stems, roots and individual leaves were assayed separately using the method of differential absorption 32 45 for computation of mixed source of phosphorus and calcium . 32 Compared to phosphorus , the delayed translocation of cal- 45 cium 'from the roots to the stem, and from the stem to the leaves, was attributed to selective exchange reactions in the transit. The proportions of phosphorus^2 and calcium^ in different parts of the plant were definitely not constant, nor did they vary in any obvious simple way. It was suggested that translocation of phosphate and calcium involves many complex processes, including mass flow as an important central mechanism. LITERATURE CITED Arndt, C. H. (1945). Temperature-growth relationship of the roots and hypocotyls of cotton seedlings. Plant Physiol. 20:200-225. Arnon, D. I. (1938). Microelements in culture solution experiments with higher plants. Amer. J. Bot. 25*322-24. Broyer, T. C. and D. R. Hoagland. (1943). Metabolic activities of roots and their hearing on the relation of upward movement of salts and water in plants. Amer. J. Bot. 30:261-73. Butler, A. W. (1953). Ion uptake by young wheat plants. I. Time course of absorption of potassium and chloride ions. Physiol. Plantarum, 6:594-616. _. (1953). Ion uptake by young wheat plants. II. "The apparent free space" of wheat roots. Plant Physiol. 6:617-35* Comar, C. L. (1951). Use of calcium^ in biological studies. Nucleonics, 8:19-31o _. (1955). Radioisotope in Biology and Agriculture. McGraw Hill Pub. 61 62 Crafts, A. S. and T. C. Broyer. (1938). Migration of salts and water into xylem of the roots of higher plants. Amer. J. Bot. 25s529-35• Epstein, E. (1953)• Mechanism of ion absorption by roots. Nature, 171:83-84. ______. (1955). Passive permeation and active transport of ions in plant roots. Plant Physiol. 30:529-35* ______. (1956). Atomic Energy Commission Rep0 No. TID-7512. A Conference on Radioactive Isotopes in Agriculture. ______. (1956). Mineral nutrition of plants: Mechanism of uptake and trans port. Ann. Rev. Plant Physiol., 7sl-24. ______and'J. L. Legett. (1954). The absorption of alkaline earth cation by barley roots: Kinetics and mechanism. Amer. J. Bot. 41:785-92. Eaton, P. M. (1955). Physiology of cotton plant. Ann. Rev. Plant Physiol. 3s 265-30 6. Preeland, R. 0. (1937). Effect of transpiration upon the absorption of mineral salts. Amer. J. Bot. 24:373-74* Gregory, P. A. (l$>37). Mineral nutrition of plants. Ann. Rev. Biochem. 6:557-78. 63 Hope, A. B. (1953)• Salt uptake By root tissue cytoplasm: The relation Between uptake of external condition. Australian J. Biol. Sci. 6: 396-409. ______and P. A. Stevens. (1952). Electric potential difference in Bean roots and their re lation to salt uptake. Australian J. Sci. Research (B) 5* 335-45. HuBer, B. (1953). Vergleichende Betrachtung der pflanzlichen saftstrome. Haturwissenschaften 40:180-85. Hylmo, B. (1953). Transpiration and ion aBsorption. Physiol. Plantarum 6: 333-405. JacoBson, L. (1951). Maintenance of iron supply in nutrient solution By a single addition of ferric potassium ethylene diaminetetra acetate. Plant Physiol. 26:411-13. ______, R. Overstreet, H. M. King, and R. Handley. (1950). A study of potassium aBsorption By Barley roots. Plant Physiol. 25:639“47. Kamen, Bo Mo (1957)o Isotopic Tracers in Biology. Acad. Press PuB. Kramer, P. J. (1949). Plant and Soil Water Relationship. McGraw Hill PuB., Kramer, P. J. (1952). The uptake of salts "by plant cells. Encyclopedia of Plant Physiology, 2:290-315. Meyer, B. S., D. B. Anderson, and C. A. Swanson. (1955)• « Laboratory Plant Physiology. 3rd edition. D. Van Nostrand Co., New Jersey. Radioisotopes. Catalog and price list, Oak Ridge National Labora tories. Revised 1-57. Steward, P. C. (1935). The mineral nutrition of plants. Ann. Rev. Biochem. 4s 519-44. ______, W. E. Berry, and T. C. Broyer. (1936). The effect of oxygen upon respiration and salt accumulation. Ann. of Bot. 50:345-66. , P. Prevot, and J. A. Harrison. (1942). Absorption and accumulation of rubidium bromide by barley plant. Localization in the root of cation accumulation and transfer to shoot. Plant Physiol. 17:411-21. Walter, L. A.,, C. L. Comar, and I. B. Whitney. (l95l)» Relative sensitive of windowless and end window counters. Nucleonics, 9:22-27. Wright, K. E. (1939). Transpiration and the absorption of mineral salts. Plant Physiol. 14:171-74. AUTOBIOGRAPHY I, Rafiq. Ahmad, was horn on September 2, 1927> in Kakori (hist. Lucknow), India. I received my high school degree from Raze College, Rampur, in 1945 and earned the Bachelor of Science degree from Muslin University, Aligarhi, India, in 1949* In 1949 I migrated to Pakistan and received a Master of Science degree in Botany from The University of Punjab, Lahore, Pakistan, in 1952. I was employed by Forman Christian College, Lahore, as a Lecturer in Botany in the school year 1952-53. Later I joined The Univer sity of Karachi as Demonstrator in Botany in 1953, and I still hold this appointment. I was awarded a travel grant from the International Institute of Education (U.S.A.) in 1956 and was granted a study leave by The University of Karachi to work for my Ph.D. degree at The Ohio State University, Columbus, Ohio. 65