ROLE OF THE ROOT TIP IN DEVELOPMENT OF ENHANCED RUBIDIUM UPTAKE IN WASHED, EXCISED CORN ROOT TISSUE By ROLANDO T. PARRONDO A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA »973 ACKNOWLEDGEMENTS The author expresses his sincere thanks to Dr. Richard C. Smith for his guidance throughout the graduate program and for his advice and help in the preparation of the manuscript. The help of Drs. R. H. Biggs, D. S. Anthony, D. G. Griffin, III, and D. B. Ward as committee members is also acknowledged. Special thanks are due to Dr. T. E. Humphreys for reading the manuscript and for making many helpful suggestions. Thanks are also due to the Botany Department for providing the author with a graduate assi stantshi p while pursuing graduate studies. Lastly, his deep gratitude to his wife Cynthia for having endured with patience the privations necessary to bring this study to fruition. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS • . ii LIST OF TABLES v LIST OF FIGURES vi ABSTRACT vili INTRODUCTION 1 REVIEW OF LITERATURE 3 Enhancement of Solute Uptake by Disks of Storage Tissue 3 Enhancement of Solute Uptake by Excised Stem and Leaf Tissue k Enhancement of Solute Uptake by Excised Root Tissue 6 MATERIAL AND METHODS 10 Plant Material 10 Preparation of Samples 12 Aging Procedure 12 Determination of K Content 12 Rubidium Absorption .... 13 Assay of Radioactivity 13 EXPERIMENTS AND RESULTS 15 Development of Enhanced Rate of Rubidium Absorption 15 Effects of Environmental Stresses on the Subsequent Development of Enhanced Rate of Rubidium Uptake 20 Development of Enhanced Rubidium Absorption Along the Apical 75 mm of the Primary Root of Corn ... 32 Effects of Aging Excised Root Tissue in CaSO. Solution and in Water Saturated Atmosphere on the Development of Enhanced Rb Uptake 35 The Effect of Time of Excision of the Root Tip on the Development of Enhanced Rb Uptake 38 Page Effects of Aging in the Presence of Excised Root Tips and in Culture Solution ^3 Effects of Concentrated Growth Solution on the Development of Enhanced Rb Uptake A8 Induction and Development of Enhanced Rb Absorption by Tray-grown and Solution- grown Roots 51 Rb Uptake and K Efflux from Excised Corn Root Segments 5^ Elongation of Excised Root Segments with Time in CaSO. Solution 5^ DISCUSSION 58 SUMMARY AND CONCLUSIONS 67 LITERATURE CITED 69 BIOGRAPHICAL SKETCH 73 Iv LIST OF TABLES Table Page I, Effects of desiccation on the rate of absorption of Rb by excised corn root tissue 26 LIST OF FIGURES Figure Page 1. Development of enhanced Rb absorption in 3-centimeter excised, tipless corn root segments aged in CaSOi^. solution 17 2. Development of enhanced Rb absorption in corn roots aged intact or aged as 3-centimeter excised, tipless segments 19 3. Development of enhanced Rb absorption in 3-centimeter excised, tipless corn root segments aged at 3°C as compared to 30°C .... 22 4. Effect of a 30-minute cold pre-aging treatment on the sdibsequent development of enhanced Rb absorption 2k 5. Effect of osmotic stress during aging on the development of enhanced Rb absorption 29 6. Effects of a 30-minute period of osmotic stress on the subsequent development of enhanced Rb absorption 31 7. Development of enhanced Rb absorption by 1-centimeter excised segments taken from three different positions along the primary root of corn 3^+ 8. Development of enhanced Rb absorption in humid air as compared to CaSO/^. solution 37 9. Effect of time of excision of root tips on the development of enhanced Rb absorption .... kO 10. Effect of time of the upper and the lower excision of 1-centimeter root segments on the development of enhanced Rb absorption .... k2 11. Effect of time of the upper and the lower excision of 1-centimeter root segments on the long-term development of enhanced Rb absorption ^5 vl Figure . Page 12. Effect of the presence of root tips, excised or attached, in the aging solution; and the effect of old culture solution used as the aging medium on the development of enhanced Rb absorption 47 13. Effect of two different concentrations of old culture solution used as the aging solution on the development of enhanced Rb absorption 50 ]k. Development of enhanced Rb absorption in root segments from tray-grown versus solution-grown seedlings ' 53 15. Development of enhanced Rb absorption compared to changes in K content during aging . . 56 vij Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ROLE OF THE ROOT TIP IN DEVELCjPMENT OF ENHANCED RUBIDIUM UPTAKE IN WASHED, EXCISED CORN ROOT TISSUE By Rolando T. Parrondo December, 1973 Chairman: Dr. Richard C. Smith Major Department: Botany "Low salt" excised corn root segments (DeKalb 805A) were used throughout this study. Rb was used as a tracer for RbCl. When ex- cised corn roots in which the apical 5 nim had been removed were washed in a dilute, wel 1 -aerated , CaSO. solution, the rate of Rb uptake in- creased rapidly with duration of washing. When root segments were washed in a water-saturated atmosphere, enhanced ion uptake did not develop during the first hour, and developed only slightly afterwards. Segments held in cold CaSO. solution, or in a solution of CaSO. and PEG-6000, or mannitol of sufficient strength to lower the osmotic poten- tial to -8 bars, did not show enhancement. However, once removed from the stressing environment and placed in CaSO. solution at 30 C, these segments developed a capacity for a higher rate of ion uptake similar to controls. A study of the enhancement phenomenon in different por- tions of the primary root revealed that this response decreased with Increasing distance from the tip, and at 65 mm from the tip It was absent. Segments taken 5 to 1 5 mm behind the tip developed the highest VIM rate of uptake. Ion uptake in these segments attained a maximum rate in 2 hours and remained constant thereafter. When the terminal 5 mm of the root was left attached during aging, but removed immediately before absorption, the rate of Rb uptake was only 28 per cent of controls in which the tip was detached before washing. When decapitated root segments were aged in the culture solu- tion in which seedlings had grown during the previous 2'4-hour period, the rate of uptake was 63 per cent of controls aged in fresh solution. Aging in concentrated culture solution was even more inhibitory to the enhancement response. When segments were incubated in fresh solution containing free-floating, freshly excised tips, the rate of uptake was 20 per cent less than in samples aged in solution containing no free excised tips. Tray-grown root segments showed a lag of 20 minutes before any increase in uptake was apparent. This lag was shorter in solution- grown roots. The evidence presented in this study shows that the en- hancement in the rate of Rb uptake with time by excised corn roots is strongly influenced by the root tip. Moreover, the results also indi- cate that this action is mediated by an unidentified substance which is synthesized by the root tip and translocated further back where it inhibits Rb absorption. ix INTRODUCTION The use of excised root tissue in mineral absorption studies is a common practice. There are several advantages of using isolated tissue or organs. The ease of handling the material makes it possible to establish better control over experimental procedures. Factors influencing the absorption of substances by the root system of plants are many. Moreover, the absorption by roots is also influenced by other processes taking place in the plant. For example, the rate of transpiration affects the rate at which water is absorbed by the roots. By using excised roots, the study of absorption is limited to those processes originating in the root itself, thus avoiding complicating effects from other organs of the plant. The advantages of using "low salt" roots in experiments measuring rates of absorption are evident when one considers that in this tissue accumulation takes precedence over translocation out of the root. In using excised roots in uptake studies, it is desirable to reduce translocation to a minimum. This is further accomplished by short absorption periods. Epstein e_t aj^. (15) listed the advantages of short-term solute absorption periods by plant tissue. However, it is realized that difficulties are encountered when trying to relate results obtained in this manner to processes taking place in intact plants in nature. Pitman (37) cautioned against assuming that the uptake by low salt roots is the same as the uptake by plants in nature. Even though the soil in which a plant is growing may be poor 1 2 in nutrients, trace quantities of these elements are present, and plants are known to "mine" the medium in which they are growing. Even though it has been known that different parts of the root respond differently to the accumulation and translocation of minerals, relatively little attention has been paid to the rate of uptake by different segments of the root. It is known that the root tip is morphologically, as well as physiologically, different from the more mature parts of the root. Moreover, it is known that hormone synthesis takes place in the root meristem, yet little work has been done to assess the role of the root tip in physiological processes taking place distal to the meristem. The root as an organ has been character- ized as being less complex than the stem. While this may be true morphologically, physiologically the root is very complex. It was shown in a recent study by Leonard and Hanson {2k) that the capacity of excised corn root tissue to absorb ions increased with time of in- cubation in a well-aerated CaSO, solution. This phenomenon was also observed in our laboratory in connection with studies dealing with recovery from physiological drought. In the present study, this in- crease in the rate of mineral uptake with time which we call the aging response ("washing" by Leonard and Hanson) was further investigated. The role of the root tip in the development of this response is speci- fically examined and its mediation by an endogenous inhibitor is proposed. REVIEW OF LITERATURE Enhancement of Solute Uptake by Disks of Storage Tissue Enhanced uptake was first observed in disks of storage tissue during aging in aerated water, or CaSO^^ solution (3, ^7). This in- crease in uptake was accompanied by an increase in the rate of respira- tion (47). Asprey (3) was the first to show clear evidence of enhanced ion absorption capacity by disks of potato and beets aged in an aerated solution. Parallel with this increase in ion absorption, there was an increase in respiration (26, kj) . Laties (22) attributed the in- creased respiration rates in aged disks of storage tissue to the development of vigorous phosphorylation. Other workers have contended that the increase in respiration is due to increased protein synthesis (2). Ellis and MacDonald (11) have reported an increased rate of leucine incorporation into proteins in beet disks with time in aging solution. The same workers have reported also an increase in nucleic acid synthesis. In a later report, MacDonald et aj_. (29) showed that puromycin inhibited the development of CI absorption capacity in aging disks of storage tissue. There seems to be a general increase in all aspects of metabolism with time. Anderson (1) stated that the aging response in storage tissue was due to the breaking of dormancy. Bryant and Ap Rees (8) summarized the aging phenomenon in slices of storage tissue as "...a reversal of changes that accompany dormancy." Enhancement of Solute Uptake by Excised Stem and Leaf Tissue More recent studies have shown that other plant tissues are also capable of increasing their capacity to absorb substances from the surrounding medium when incubated in well-aerated solutions of vari- ous salts or water. Bieleski (5) isolated vascular bundle tissue from celery petioles and aged it in CaSO, solution for 20 hours. After ^ to 10 hours of aging, the rate of phosphate uptake rose to 50 times that of fresh tissue. The sucrose accumulation rate was 3 to 8 times that of fresh controls. Bieleski suggested that the low affinity uptake mechanism of solute absorption was present in fresh tissue, while the high affinity mechanism developed during aging of the tissue. Hancock (13, 19) arrived at the same conclusion for 3-0- methylglucose (a non-metabol izable derivative of glucose) absorption by squash hypocotyls. The formation of the high affinity mechanism was not affected by light conditions, shaking, or the absence of CaSO, from the solution (<19)- A typical response curve obtained in these tests can be described as a lag of 2 hours, then a rapid in- crease in uptake up to 12 hours followed by a constant rate thereafter. Palmer and Loughman (3'*) have reported that incubation of pea stem segments in potassium maleate buffer caused an increase in the rate of phosphate absorption. At the same time the rate of respira- tion decreased. Cotton and sunflower stem segments incubated in the same medium failed to show any increase in the rate of phosphate ab- sorption. They showed a doubling in the rate of Rb absorption by pea stem segments after 18 hours of incubation. In a later study, Palmer and Blackman (35) reported that the enhancement in phosphate uptake which developed during washing could be prevented by the addition of 5 2,4-D to the washing solution, Leonard and Hanson (2^) confirmed the inhibitory effects of Z.^t-D and lAA on the development of enhanced phosphate uptake. However, they also reported that washing in the presence of 2,4,6-T, a substance which has little auxin activity, was even more inhibitory. Rains (41) noted an increased absorption of K by bean stem slices aged for 20 hours in CaSO/^ solution at 30°C. He showed that this enhancement in K absorption was prevented if the tissue was incubated at k C. Addition of benzyladeni ne (an analog of kinetin) to the ab- sorption medium inhibited the enhancement of K absorption. The same effects of benzyladenine have also been shown in aging disks of tobacco leaves (M+). In a later report. Rains and Floyd (42) reported that the increased K absorption rate in aged bean stem slices was promoted by Ca. Aging of bean stem slices in the presence of cyclo- heximide prevented the enhancement of K uptake, thus indicating that inhibition of protein synthesis also prevented the increase in K up- take developed during agirvg. The process controlling the increased ion uptake in bean stem tissue appeared to be different from the process in storate tissue in as far as there was no increase in the rate of oxygen uptake. On the contrary, respiration decreased during the first k hours of aging (16). Macklon and Higinbotham (30) reported a considerable increase in K and NOo content in excised segments of pea epicotyls immersed in complete nutrient solution for 72 hours, as compared to intact, etio- lated tissue. During this time, there was an increase in cell electro- potential differences. Although the increase in ion uptake showed a lag of 6 to 8 hours, the difference in cell potential increased 6 rapidly. They considered the increase in potential difference a pre- requisite for the rapid ion accumuJat ion. Incubation of sufficient duration in water, prior to placing in solutions containing K, elimi- nated the lag in K uptake. Sacher (^3) reported an increase in up- take of orotic acid, glucose, and phenylalanine from 5- to 50-fold in bean endocarp after aging the tissue in water for 2k hours. Auxin (NAA) largely prevented this enhancement in substrate uptake. Enhancement of Solute Uptake by Excised Root Tissue Enhanced solute uptake has also been reported for non-dormant, excised root tissue. Tanada (48) using segments of mung bean roots showed an enhancement in Rb uptake with time by samples incubated in solutions containing Ca. No increase in uptake was shown when Ca was absent. In the presence of Ca, the uptake of Rb was not immedi- ately stimulated. There was a lag period of about 10 minutes before the rate of absorption gradually increased and then proceeded linearly at a rate much faster than the initial rate. In mung bean, the enhance- ment of Rb uptake was most pronounced in the 2.5 to 5-0 mm zone from the root tip. Tanada attributed the enhanced Rb capacity to effects of Ca on the availability of binding sites in the membrane-bound carrier. The role of Ca in ion transport by plant tissue has been emphasized by Epstein (13)- Cereals, particularly barley, have been favorite plants for mineral uptake studies. Pitman (39) reported that when excised barley roots were kept in 0.5 mM CaSO, solution for 2 to 4 hours, this tissue took up more K than either intact plants or freshly excised roots. He attributed the net increase in K uptake to an increased selectivity 7 of K over Na, and to increased K efflux from the tissue. However, no difference in tracer uptake was reported. In a recent study Pitman et aj[. (40) reported on the changes in electropotential dif- ference between excised barley roots and the dilute CaSO/. solution in which they were aging. After 6 to 8 hours of aging, the potential difference increased from 65 millivolts to I85 millivolts. As pre- viously reported, there was no difference in tracer uptake between fresh and aged tissue. They suggested 2 possible hypotheses to explain this effect of aging on potential difference: (1) that cutting exposed plasmadesmata which are leaky initially, but which seal in time, and (2) that some internal factor, such as hormones, have a regulatory effect on cell potential; an influence which dissipates with time after excision. Leonard and Hanson {2k) reported a I50 per cent increase in the rate of phosphate absorption by barley root segments after 3 hours of washing in aerated 0.2 mM CaCK solution, and a 200 per cent increase in phosphate absorption by oat root tissue incubated for the same period of time under the same conditions. There are several reports of increased rate of mineral absorption in excised corn root tissue. Brown and Cartwright (7), using apical segments of corn roots in which cells were not fully elongated, showed that increased Rb uptake was due to higher protein content in expand- ing cells. Handley et a_[. (20) using segments of corn root tissue taken 1.8 to 3.8 mm from the root tip, showed that aging increased the rate of Rb uptake over freshly excised tissue. When pre- incubated for 2 hours in CaCl2, the rate of absorption remained constant. They attributed this phenomenon to a dual effect of Ca on Rb uptake: an initial inhibitory effect, followed by a stimulation with time. A 8 similar effect of Ca on the rate of K uptake in corn root tissue, but not in barley, was reported by Elzam and Hodges (12). Laties and Budd (23) reported a 20-fold increase in CI uptake by aged corn root steles over fresh steles, while the increase in aged cortex tissue was only 2.5 times that of freshly separated cortex. They attributed this difference in absorption between fresh and aged steles to a leaky condition of freshly isolated steles. That some stelar cells are normally leaky was proposed earlier by Crafts and Broyer (10). In a later study, Luttge and Laties (27) reported a 10-fold increase in K absorption by aged corn root steles. They con- cluded that this increase in uptake in aged steles was due to develop- ment of a high affinity mechanism of solute uptake (14). Yu and Kramer (^9,50 ) reported a higher content of Rb in excised corn root tissue than in intact roots after 23 hours of absorption. However, these workers were not able to confirm the early findings of Laties and co-workers on the increased capacity of uptake by aged, isolated steles over the cortex. in a recent study. Hall £t aj^. (17) reported a greater increase in the rate of CI uptake by isolated steles than by cortex of corn roots, after 2k hours of aging. However, the in- crease in uptake by isolated steles was much less when the seedlings were grown under sterile conditions. Leonard and Hanson {2k) have reported a 280 per cent increase in the rate of phosphate absorption by excised corn root tissue that had been aged in 0.2 mM CaCl2 solution for 3 hours before absorption. This enhanced rate of uptake was prevented by anaerobiosi s, low temperatures, metabolic inhibitors, and several plant hormones. An enhanced uptake capacity appeared to be general, since the rate of 9 absorption of several substances was also increased during 2 hours of washing in dilute CaC^ solution. The presence of Ca in the washing solution was not necessary since incubation in distilled water gave similar results. After a lag of 30 minutes, the tissue showed a constant rate of increase which continued for k hours before leveling off. Analysis of membrane-bound ATPase showed an increase with time in washing (25). Electron microscopy of subcellular structures of fresh and washed tissue showed no detectable changes due to washing. Since efflux of ions has been reported to take place readily (21, 31, 36, 38), some workers have attributed the increase in absorp- tion to the loss of minerals during aging (39). However, Leonard and Hanson (2^) reported net accumulation of K after the tissue was washed at 30 C, but not in freshly excised tissue. Handley e^ aj.. (20) showed no change in K content in corn root tissue after 6 hours of aging in solution. The influence of microorganisms on the development of enhanced solute uptake by excised tissue has been reported by several workers. Palmer (33) and MacDonald (28) have shown that in storage tissue, non- sterile conditions reduced considerably the rate of uptake. However, Bowen and Rovira (6) reported that uptake of ^^P by tomato and clover tissue was higher when plants were grown in non-sterile conditions. Barber and Frankenburg {k) reported rates of uptake of phosphate and Rb by excised barley roots to be higher in plants grown in non-sterile conditions than in plants grown under sterile conditions. Leonard and Hanson {2k) aged corn root segments in the presence of chloram- phenicol and found no difference in the rate of phosphate uptake be- tween control and treated samples during 8 hours of incubation. MATERIAL AND METHODS Plant Material Solution-grown Roots The tissue was obtained from the primary root of ^t-day-old seed- lings of Zea mays (DeKalb 805A) grown according to the method de- scribed by Smith et aj_. {k6) , but slightly modified. One hundred grams of dry seeds were placed in a 2-liter Erlenmeyer flask contain- ing 1 liter of distilled water, shaken and the water drained. This washing procedure was repeated twice. The seeds were surface ster- ilized in 200 ml of 15 per cent Clorox (5 per cent sodium hypochlorite) to which a few drops of liquid detergent were added. After 5 minutes of gentle shaking, the detergent-Clorox solution was poured off and the seeds rinsed with distilled water 10 times. In the last 3 rinses the flask was allowed to overflow. The flask containing the seeds was filled to the I8OO ml mark with distilled water and the seeds were soaked 18 to 20 hours with vigorous aeration. Then the seeds were rinsed 3 times with distilled water, and the flask was filled to the 1800 ml mark with distilled water and placed in the incubator. After 2 to 3 more hours of soaking, the water was decanted, and the seeds rinsed with distilled water. At this stage most of the seeds showed the coleorhiza protruding through the seed coat. Seeds not showing the coleorhizB at the end of the soaking period were discarded. The remaining seeds were planted between 2 layers of boiled, coarse-grade 10 11 cheesecloth. The cheesecloth was supported by a stainless steel mesh and hung at the top of a if-liter beaker filled with 0.2 mM CaSO. solution. The seeds were distributed among k beakers. A sintered glass aeration tube was placed inside a glass jacket and this assembly placed in each beaker for aeration purposes. The corners of the cheesecloth were placed to wick the solution, but the seeds were held a few mm above the aerated solution. The beakers were covered with a watch glass and placed in the incubator. After Zk hours the top cheesecloth was removed and discarded and the seeds washed thoroughly with distilled water. At this stage most of the roots were about 2 centimeters long and were extending into the solution. The seeds were placed over fresh 0.2 mM CaSO/^^ solution in a clean beaker, the watch glass replaced, and the beakers placed back in the incubator. The third day the same procedure was followed, except that the watch glass was not replaced because of the length of the coleoptiles. The roots were harvested the fourth day. At this stage the roots were usually 12 to 15 cm long. Tray- grown Roots The seeds were sterilized as described above. After the tenth rinse with distilled water, the seeds were planted in Pyrex trays, with the embryo side in contact with several layers of white paper towel saturated with 0.2 mM CaSO/, solution. The trays were covered with transparent food wrap in which holes were punched to allow gas exchange. The trays were placed in the incubator at 28 C and left undisturbed until harvest time. The roots were harvested the fourth day. At this stage the roots were usually 8 to 10 cm long. 12 Preparation of Samples Root segments of either 3 cm or 1 cm, taken 5 mm from the root tip, were the experimental material. Immediately after excision, the segments were placed in a small fiberglass basket in a 2-liter beaker containing 0.5 mM CaSOi^. They were then agitated for I minute to remove material from damaged cells, removed, gently blotted, weighed to - 0.1 mg, and placed in fiberglass bags for handling. Each bag of 10 or 15 root segments was prepared separately and as rapidly as possible, requiring 6 to 8 minutes. Aging Procedure The aging solution consisted of k liters of 0.5 mM CaSO^^ solution prepared in if-liter beakers. The temperature of the aging solution was maintained at 30°C in a water bath. The solution was vigorously aerated throughout the aging process. Hereafter these are referred to as standard aging conditions. Seven or eight samples, each con- taining 10 or 15 root segments were aged in each beaker before dis- carding the solution. Determination of K Content Samples used for K content determination were rinsed 3 times in distilled water after aging, placed in crucibles and ashed at 500 C. The ashes were dissolved in distilled water and diluted to exactly 25 ml in a volumetric flask. The K content was determined by using a Flame Emission Spectrophotometer (Beckman B and DU) . 13 Rubidium Absorption Preparation of the Solution The absorption solution consisted of O.I mM RbCl and 0.5 mM CaCl2, prepared in a 2-liter volumetric flask. Enough ^^Rb was added to give a counting rate of approximately 10,000 cpm per pmole of RbCl. Four-hundred ml volumes were poured in 500 ml wide mouth Erlenmeyer flasks and placed in a water bath at 30°C. The flask con- tents were aerated vigorously throughout the absorption period. At the end of 10 minutes of absorption, the concentration of Rb in the solution had decreased less than 1 per cent. Uptake Procedure Thirty seconds before absorption started, each sample was removed from the aging solution, and swung around to remove excess water from the bag. At exactly zero time the sample was dropped in the absorp- tion solution. After 10 minutes, the absorption of Rb was stopped by dropping the sample in cold (3°C) exchange solution, containing 5.0 mM KCl and 0.5 mM CaCl2. After 3 rinses, the samples were submerged for 30 minutes in k liters of aerated exchange solution kept at 3°C. At the end of exchange the samples were rinsed 3 times in distilled water and the tissue placed in stainless steel planchets for determination of radioactivity. Assay of Radioactivity The [ilanchets containing the samples were ashed at 500°C. After cooling, the ashes were dissolved in water, and a drop of detergent solution added to break the surface tension. The samples were evap- Ill orated to dryness under low heat on a hot plate. The activity of the samples was determined by a low-background counting system in which the sample detector was a G-M tube with window thickness of 150 pg/cm^. The background averaged less than 2 cpm. Each sample was counted at least 2 times to 10,000 counts, or for 10 minutes, which- ever came first. Each experiment was repeated at least 2 times with similar results. The data shown, however, are from single experiments EXPERIMENTS AND RESULTS Development of Enhanced Rate of Rubidium Absorption When 3-cm segments of corn root, in which the terminal 5 mm of the tip had been removed, were aged in well-aerated 0.5 mM CaSO. solution, the rate of Rb absorption increased rapidly (Figure I) for several hours until it reached a maximum and then approached a con- stant value. In some experiments, however, after reaching a maximum, the rate of uptake declined. This was attributed to a depletion of food in the excised tissue. Figure 1 shows that after 8 hours of incubation, the rate of absorption of Rb was 13 times that of freshly excised tissue. To test whether the development of the enhanced rate of absorp- tion was due to excision, an experiment was designed in which one group of roots were excised and aged under standard conditions. In another group, intact roots were incubated in 0.5 mM CaSOr at 30°C, and then segments excised before determining the rate of absorption. At different periods of time after aging (incubation) started, a sample from each treatment was taken out and its rate of Rb uptake determined. The results of this experiment are shown in Figure 2. In both groups the capacity to absorb Rb was Increased. However, the rate of absorption was greater in roots which had baan axcitad. Excision amplified this response, but was not the cause of it. Six hours after transfer to fresh CaSO^ solution, segments from roots 15 T3 Q) U> 10 M 4-1 C v E cn 1 0) lA 4-> O O U c L. o u • 1/1 z in E 0) p— LTV a. • ,— o +J in * m T3 3 0) U) c •_ o O X 4-) 0) ZJ ^^ i- o 0) tn 4J 0) cn E c 4-) Ol c TO o u U 1 -C cr\ -l-l C M- — o c C o o • ^ 4-> +J Q. (0 1- 1- o 4J U) c -Q d) o cx: u ■D 4) 0) x: (J 1- c TO ^ • c c 0} o **- 'M o 3 rM> 4-1 o C in ^ -^ D.O O in ^— (D V <_> > V C Q >« o 1. 3 17 C/5 o LlJ < 00 r- CO Lo "Ni" ^ c\j — .,.•♦••• (uiuu 01 ' ^/sd\o\i}rl) uoijdjosqv qy (U c *-> (U u 1 8 po a: «i (D • -3- ■D o 0) I/O Ol (D flj (-> 1- S O E +J UA u (D O 4J c <+- — O XI -o 0) (U C7) +J (D tf» W) tn 4-> c O o O u U c c o 1- o 4J u 3 c "o — in c en o c • 4J en Q. Q. (0 • a. u +J o 0) w JZ 0) J3 H ^ (D ■I-) ja • £ cc tf) Q -M t- -D C <4- (1> (U o E E c CD E OJ u 1- ♦- •M 0 -TD T) 0) 0) -M tn (rt • — •— u «n X c tt) 0 0 i_ V c 4-1 0 4) ._ E *-> — 3 4-> — c 0 0) 1/1 u • 1 01 Q. rr> C •— ■— *J C Ol .- HJ V s: C 0) 4-< 0 ^ — 1- E *-l 0 Q. L. L. • >*- 0 0 u) 0 E X) 0 E (D P^ Lr\ -n 0 Cf\ oc ■•-> 0 TJ -D 4-> 0) 0) 0 1- Lr\ C (» -D E > 0) cn OJ CD 0) O (Q (/) CO 0) u 3 cn 22 O O o ^0 o o O CD < CO < } ) L - -sT - CM O X LU (3 CD < O h- CD LO '^ ro CM — » • o ■ a » • (•uiuj 0! ' &/S9iotur/) uojjdjosqv qy "O 0) -a 4-> (U I/) o . c w +J 3 c ft) "o ^ I/I (i o ai 1 • c a. 0) •^ •.. > CD +J 0) CO -D 0) 0) -C *-> ^ +J c 1- a> E 3 O cr • 1- 0) -*4- w O J3 CO E D (D E (/> u ir\ 0) z cr\ jr E 4-1 O LA 4-» C • O O lA ■M »4- C C o 0) 0) J^ E TJ 0) 4-' (/) V k. •— . l_ •M 1/) 0) C s CT o C u in •_ 4-> 01 c C (D o x: 3 0» t- C s: •^ I \- E 1 • -3- o • ' o <*\ c CO o ^ OJ (D 4-i c_> O Q.: o i 4-1 in 1 LA o . J3 • 0) <0 1 O u- M- . H M- UJ OC O • 4 9 O) 2k CD f^ CD ID ■^^ ro (M — • • • • • • • • • • (•uiiu 01 • 6/seiouLiT/) uojidjosqv qy 25 Effects of Water Stress Drought conditions were simulated in the laboratory in two dif- ferent ways: by desiccation and by incubation in solutions of low osmotic potential. Excised root segments were suspended over 500 ml of 5.0 M NaCl in 2-liter flasks where they remained for various time periods. Control samples were suspended over distilled water. The flasl 0) SI in y- 3 1/1 • +J c c O 0) — E ■!-» O) Ql 0) I- VI o sx o OJ o Si SI •o h- — ■a c E O 1/1 — t. «- o E +-> 00 E \n (0 4J O O O +J E 1= in O lA O — 4-> C O t- -^ 4-* lU +J C +-• U 0) >. o» CM CVI + d^ O^ (J) CO a a O O k \ ro o LU < HO CD 00 r-- CD LO 'sj- i^'^ CO - (•uiijU 01 • 6/s9|ouu77) uojidjosqv qy x> t) u LTV c m c j: a> c j<: V y- o +J (U c 3 0) e in a. +J o c 0) "oj E > cn 0) 0) TJ O 3 L. cr 0) u -tj +j 3 O +J »*- c o -D tt) O 1- Q. L. (U (/» Q. s V • ■t-i ■4t3. 3 O •^ C C/) +J •— nj E o 0} O +J cr\ • c E (D o O 1. U- +J ^ O Q. 1- E U) O E +J in o Xt LA (U (0 ra I+- M- x> O UI oc ■M • vo » 1. 3 o> 31 f" CX) 0) o5 o o o to I o LJ CL c o I o O c o I o Q- O irr -— o o ro ro to LjJ o CD < C\J ID O CO h- CD LO ■^ ro cvi (•ujuj 0! • &/saiouuT/) uoudjosqy q>j 32 by this osmoticum. It is not known whether this damage was due to direct toxicity or due to damage to membranes or other subcellular structures. Development of Enhanced Rubidium Absorption along the Apical 75 mm of the Primary Root of Corn This experiment was conducted to determine to what extent the enhanced capacity of Rb uptake developed at different distances from the apex of the primary root of corn. Due to the presence of lateral root primordia near the base of the root, this study was limited to the apical 75 mm of the root. One cm segments were taken 5-15, 35_Z+5^ and 65-75 mm behind the root tip. Each sample contained 15 root segments with an average weight of 120 mg. These segments were aged in 0.5 mM CaSO/^ for periods up to 9 hours before measuring the rate of Rb uptake. The results of this experiment are presented in Figure 7. Segments taken 5 to 15 mm behind the root tip showed the highest increase in the capacity to absorb Rb. The maximum rate was reached in 2 hours, and remained constant thereafter. The basal segments had the highest initial rate of absorption (0.31 fJmoles/g- 10 min.), and this rate was not affected by the aging treatment. The middle segments, which initially absorbed at the same rate as the apical segments, increased their rate of uptake to the level of the basal segments, but did not go any higher. This experiment was re- peated 3 times, with similar results being obtained. On the basis of these results, all subsequent experiments utilized only the apical segment, 5 to 15 mm behind the root apex. c tn a> ■ ^ ^ U) m C 4J 0 u (rt ■M c C o cn 1 3 0) ^ ul o U1 T3 0) in irt c U cn X ID 0) (U l_ -C (U H- +J (U E • c ■M u C o V u o 1 M- (U o c o 4-1 o >- o -Q 1- c >- O 1. (0 4-1 ^ a. .. 1. l- o a. w JD J3 cr: U) c XI o 0) u ^ c (C v> ^ c c o 0) ' 1^ ^ I*- . •■* o v> o 4-1 Q. c 0) 4J • E c -a- Q. 0) O O u Ln V > M- dJ . - z: Q -D e • r«- o ot 34 CO £ e E £ £ E in LO 1 LO 1 I 1 ID ro 1 in H CD to LO Z3 o X hi ^ h- K) o < CO o i CD CD r- CD LO '^ ro c\J " {'ui'jjoi ' b/seiOOiT/) uojidjosqy q^ 35 Effects of Aging Excised Root Tissue in CaSO; Solution and in Water Saturated Atmosphere on the Development of Enhanced Rb Uptake The results of Table 1 show that segments placed over distilled water developed only a small increase in their capacity for Rb uptake with time. This was further investigated. One-centimeter segments were aged in a water saturated environment or in aerated 0.5 mM CaSOK under standard conditions. Experimental samples were placed in 2-liter Erlenmeyer flasks containing 500 ml of distilled water. The flasks were sealed with Paraf i Im and placed in a water bath at 30°C, 2k hours before the experiment started, as described before. One sample was placed in each flask. To insure that the tissue did not suffer any significant loss of water, the fiberglass bag containing the tissue was covered with a piece of damp cheesecloth. In the flask, the edges of the cheesecloth touched the water, but the bag containing the roots remained several centimeters above the water. After hanging the sample inside, the mouth of the flask was sealed immediately with a new piece of Paraf i Im. The oxygen in the flask was calculated to be 300 times that consumed by respiration of the tissue during a 4-hour period. The results of this experiment are shown in Figure 8. Samples aged in CaSO^ solution showed the usual enhancement of Rb uptake with time, reaching the maximum rate in 2 hours. However, little enhance- ment of the absorption capacit!y occurred in experimental samples held over distilled water. After 1 hour, the rate of uptake had not changed. After 2 hours, the rate of uptake was only 15 per cent of controls. A sample taken out after 2 hours of aging over distilled water and immersed in aerated CaSO/^ solution for 1 hour showed an increase in the rate of absorption similar to controls aged in CaSO/^ solution o o 1_ o o o CO (D • O Q. -o O (U O L. I. (D Q. 0) e ^ o •M u E w O (0 1. u- u *nj i TJ lA • «• w^ E 3 O C UA c c a> o j^ (0 *+-! ■M Q. i- ^ O O) U) c x> o (0 XI u a: a> +j T3 0) «) E U C 4-> (D C x: 0) c u 0) ^^ u- o 0) L. •M 4) C 3 0) E 10 Q.4J O c » "« E > cn 0) o o in 00 0) I. 3 cn 37 'n1 4.0 O r ooo- T \ co o / \ v-<^ / \ Wate Into / \ c? ^ - E§ / \ o o< / \ O ^-o x.i=- / \ ■- ---5 cvj< "O Q / \ \ < <(f) < 3 1 \ \ / ^x V ^ ^ / "^ \ / **^ \ / \ \ i \V \^1 ^a t 1 1 1 1 ! 1 t ro CO 10 ^- 13 o X (^ CD (•UlLU 01 uo "^j- ro cvj — • • • • • &/S9iOUJTy) uoijdjosqv qy 38 throughout the aging period. Changes in weight in experimental samples were less than 1 per cent and in some cases the tissue gained weight. The Effect of Time of Excision of the Root Tip on the Development of Enhanced Rb Uptake Apical root segments were aged with and without the root tip attached, but the root tips were excised in all cases before Rb ab- sorption. Figure 9 shows the results of this experiment. The presence of the root tip during aging largely prevented the enhancement of Rb absorption. Segments aged without the tip rapidly increased their rate of uptake, most of it taking place within the first hour of aging. At the end of the first hour of aging the rate of uptake by experi- mental samples was only 10 per cent of controls, and at the end of 8 hours of aging the rate was still only 2k per cent of controls. In order to determine further the role of the root tip in prevent- ing the enhancement of Rb uptake, another experiment was designed in which the roots were aged under k different experimental conditions. (1) Roots excised and aged with the terminal 5 mm of the root intact during aging but removed immediately before absorption. (2) Roots treated as in 1, except that the terminal 5 mm of the root tip was removed before aging. (3) Root tip (5 mm) removed, the seedling incubated in 0.5 mM CaSOr, and root segments excised just prior to Rb absorption, (h) Intact seedlings incubated in a culture solution in which seed 1 ings had grown for 2k hours before the experiment, and samples excised before Rb absorption. At different time periods a sample from each group was taken out and the rate of Rb uptake deter- mined. The results are shown in Figure 10. As before, excised segments *J • o ^ o o en CO 10 c z o E 4-> Lf> a. . t- o o U) u- x> o ID ■o J3 0) • ac +J -o U1 o TJ • — 0) V) 1- u c 0) c o Q. (0 o JZ cn c c c 0) o •^ o> M- •M ID o 3 ^— 0) +J o j: c (rt 4-1 (U E Dl L. Q. c 0) o • « 4-1 CD It- "Z ID ID > V lU ■o TJ s: a> H > 0) o ^ E 4-) • 4) Q. l- C •^ O ■M ID U) 0) 3 Q. j: •^ 4-1 Q. -M ••» £ 4J ■M o O u 4-> O <4- O I. O E u U- E o 0} in Ji c ^— 4-> o •^ o •« M +j in — 0) u LA X Q. 0) C E lU ID »4- -^ (/> o ID 4-> . ^M V ID B V 4-> u C ■M 0) 0) v^_ 3 E o U1 1. 4J V 4-) c Q. u 0) X (0 >4- E 0) H- 0) c UJ in • ■^ s 3 on 40 O - ^ LlI < en • — in C o 3 W1 urv a +J .^ c c ■t-> 0) 1) E j^ 0) cn 1 (D ^ tt) ■M 4J i/i 0) 4-J k- ■D o a> V o 3 Ji L. O 1/1 ro u •M ■u 0) C 4J +J 0) C 0) -M 0) o O j: 1 o -M ^~ I. j: M- 0) ■M o JC • ^ H- 3 c o ■D •— • 0) tf) c cn •« o (D o X +J 4) a> Q. u L. 4) i_ o 3 0) in 3 JD irt o Q. 3 Q. Q.U- 3 O in cn 4) c (U +J I/) •-• JZ c » JZ *j (U 'Z e > • k- V o. 4) +-> -o •— ■M ■M ' 14- i*- E (D o u 4) (U x: ' ■D 4-> +J •M 4) u 1 > 0) cn ^ O M- n Q E U- o 1- 4) UJ 4- 1- 45 en c o> o o» c u. zy •o TJ c> JZ o o Q. J: •*- o O Q. .d <*— ■♦^ "s r/> O x: o n> ♦" c x: •—• ■ 1 — •^- 5 -o o 3 «. G) T3 CO T) O) O O ■>— t/> o O n o X X UJ i LJ ± ± 1 ±. ± CVJ C\J CO O (M CO if) JS^ 6 OJ UJ H o cp Hco§ CD - ^ - CVJ o CDOOh- (DLO^rOCVJ — {•uiuu 01 • 6/S910UUT/) uoijdjosqv QH 0) -C +J ■D •h 0) TJ "2 c c +J c i) +J o ID ._ <4- •I-J O Q. 3 •^ •■i— 4J ■u o C (« 0) lU E x: CD Q. +-> C O ... -C ai lU 4J (D > ,—. V 3 c 4-* i_ CM 4-1 c 0) O o 3 0) O .— X u 4J i/i +J D 0) It- f— 1_ 0 O "a. 0) w E 4-> (U (D M- o 0) U1 < > l_ 3 O Q. U E • lU 0] ■o C t- x: ^— o 4-> o • ^ tn +J ID U- H- Q. 3 O o 1- O a. ■M ■M in •.. u u X) ■M 0) V (D >+- >+- 0) >+- M- J3 X LU 0) o: *J CM 0) u 3 D1 47 sz I/) CD i CD -C o a D Q. CD v_ CO O .S!2 X c o o CI 5 .E en D O X c o o V) c "en o CO c:l •o CD CO O X CD CO o o to o X CU CM OJ OJ XJ 1 D T) "O CD CD CD (D cn cn D^ cn < < < < ■ 1 -1 • • m 9 1 • « ■ ;.« I'-:':-:' • •V i<>< L ZJ a O ■D • t> CNJ c en o 3 13 0) 4-1 C Ol 1 • — •M 0) >— . 3 u ■ .. p^ (U s: o 5 a. u» o >« 4> 0) L. 3 Q. >- +J E J3 ^-> IB 3 lO ^^ U E -o . o — C o o o LTV u- J-> o o Q. I. 4-1 U) O 1- c tn 0) o ^ 4-> • — > m •— 4-1 ^ *■> c (D 0) H- L. L. o 4J 0) C >4- 4-> V U- C (J •— 0} c tJ £ o a o Q o 5 1 ^M Irt ■M 0) (0 > 3 >4- 01 . o x: 4J 4) 4-> 3 M- ■a M- C O UJ o M f<\ I. 3 Ol 50 c o o (f) 5 o CD CD O c o VI 10 D • m Q. u • ^ 4) +J > ■M c o 5 o O 1- u CTl (U 1 j: >.-M 0) O ^ o +- U) o c ■!-> ^_ C ■D 0) (0 E (U Q. w O r^ c 0) s > o 0} L. o cn !» 3 53 (^ CD in 'sT f^ CM — ; • ••••* (uiuj 0! - &/s9ioujTy) uoi^djosqv qy 5^ Rb Uptake and K Efflux from Excised Corn Root Segments The following experiment was carried out to determine the extent of K loss from corn root segments and the degree to which this loss is influenced by aging. Excised root segments were divided into 2 groups. Both groups were aged under standard conditions. Two samples were taken out at each sampling during aging. One sample was used to determine the rate of Rb absorption, and the other sample was used to determine the K content. The K in the roots would necessarily have to come from the seed since K was not supplied at any time during the growth of the seedlings. The results are presented in Figure 15. The data show that the K content of the tissue remained constant during 8 hours of aging. The enhanced Rb uptake with time was not accompanied by an increased efflux of K from the tissue. Although K efflux might be significant in tissue with high salt content, tissue with low salt content such as that used in these experiments did not lose significant quantities of K during aging in CaSO, solution. Elongation of Excised Root Segments with Time in CaSO; Solution To determine whether further elongation occurred after excision and during aging, the change in length of 1 -cm root segments, taken 5 to 15 mm behind the root tip was checked. These segments were accu- rately cut with the aid of a stereoscopic microscope. After 4 hours of aging in CaSO^ solution under standard conditions, the increase in length was less than 1 mm. Thus It can be assumed that most of the cells in the segments had completed their elongation, or conditions for further cell elongation were lacking. Therefore, the enhancement 0) JZ t- « en c en 03 en c 1- D -D 4-1 C 0) • ■M Q. c • « O +J u 4J a<: O o c L. V (rt j: 0) *j en c E m O .c i_ u VI- o E 4J E ■D LTV « ^ L. (D o Q. ■4-> £ Q lA o C c t) o ^ (TJ •t-l +J Q. L. •^ o Dl W1 C J3 o (D — J3 1- o: 4) 'M ■o « 9> E u c 4-1 10 C -C o c o 0) ^— M- o 0) u •M 0) c 3 o E in Q.-t-* O c ^ 4) 0) E > CD v 0) o in lA £ 3 (jM qsajj 5/Blu) luaiuoQ y\ CD C\j CO 'sT 56 CO h- CD LO ^'-^ ro c\J — (■Ujoi 0! • 'o/SQ\o\i\rI) uo!|djosqv q^ 57 in the rate of Rb uptake of such proportions as shown in this study could not be attributed to cell growth. DISCUSSION There have been scattered reports in the literature (20, 2k, 48) of a time-dependent enhanced capacity for mineral uptake by excised roots. Many of these reports have been limited to isolated observa- tions made in the course of other studies relating to mineral absorp- tion. Leonard and Hanson were the first to publish the results of experiments aimed at determining the causes of this increase in uptake. At the same time this phenomenon was being observed in our laboratory. It is of particular interest since so much work has been published on the subject of mineral absorption using excised roots of grasses. A clear understanding of this phenomenon is needed to relate uptake by excised tissue to intact plants. In this study an attempt has been made to determine the nature of this enhancement, as well as the extent of its development along the primary root. An effort has also been made to determine the cause or causes which induce this enhancement and the locus in the plant where it originates. The mechanism of increased mineral uptake in corn root tissue does not seem to be the same as that operating in slices of storage tissue, the time dependence curves are different. In storage tissue, a lag time of Zk hours has been reported before an increase in uptake was apparent (^7) . in excised, sol ut ion- grown corn roots an increase in uptake was evident after 10 minutes of aging. In tray-grown roots, a lag of 20 minutes was required before increased uptake could be detected. Leonard and Hanson reported a 30-minute lag in excised corn 58 59 roots (24), In corn root tissue the increase in uptake reached a maximum i n 2 to 4 hours, while in potato tuber slices the rate of phosphate uptake was still increasing after 30 hours of aging (26). The increase in uptake by storage tissue is accompanied by an increase in respiration, typical of tissue breaking dormancy (26). In corn root tissue the rate of respiration remains constant (24). The enhanced rate of Rb uptake was not considered to be due to microbial contamination. Even though no attempt was made at determin- ing the bacterial count in the aging solution, there is evidence which indicates that microorganisms were not responsible for the enhancement. If microorganisms were responsible for increased uptake, the rate would continue to rise as the bacterial population continues to increase. However, the rate of Rb uptake reached a maximum in 2 hours and remained constant thereafter. Further evidence published by others (24) showed that incubation of corn roots in the presence of chloramphenicol at bacteriostatic concentrations did not have any effect on the develop- ment of enhanced mineral uptake capacity. The response of Rb uptake to aging could not be attributed to increased K efflux from the tissue. The K content of excised roots remained constant during 8 hours of aging in CaSO. solution under standard conditions. Similar findings have been reported for phosphate uptake by Leonard and Hanson (24), and for Rb uptake by Handley et a1 . (20). Whatever the cause, or causes, it appears that the increase in uptake is dependent on the metabolism of the cell. This is Indicated by th« fact that when excised roots were aged at low temperature at which metabolism is minimal, the enhancement of Rb uptake was reduced 60 (Figure 3), or prevented {2k). However, when the tissue was returned to 30 C the enhancement proceeded rapidly (Figure k) . Further evidence which indicates that this process is metabol ical ly dependent is given by results from water-stressed tissue. In tissue under water stress, the enhancement of Rb uptal- kkO. 26. Loughman, B. C. I96O. Uptake and Utilization of Phosphate Asso- ciated with Respiratory Changes in Potato Tuber Slices. Plant Physiol. 35:'»l8-'»25. 27. Luttge, U., and G. G. Laties. 1967-. Absorption and Long Dis- tance Transport by Isolated Steles of Maize Roots In Relation to the Dual Mechanism of Ion Absorption. Planta 7'»:173-l87. 71 28. MacDonald, I. R. I967. Bacterial Infection and Ion Absorption Capacity. Ann. Bot. 31:163-172. 29. , J. S. D. Bacon, D. Vaugham, and R. J. Ellis. I966. The Relation Between Ion Absorption and Protein Synthesis in Beet Disks. J. Expt. Bot. 17:822-837. 30. Macklon, A. E. A., and H. Higinbotham. I968. Potassium and Nitrate Uptake and Cell Transmembrane Electropotent ial in Excised Pea Epicotyl. Plant Physiol. '♦3:888-892. 31. Marschner, H. , R. Handley, and R. Overstreet. I966. Potassium Loss and Changes in the Fine Structure of Corn Root Tips Induced by H-ion. Plant Physiol. '♦1:1725-1735. 32. Michel, B. E. 197'. Further Comparisons between Carbowax 6OOO and Mannitol as Suppresants of Cucumber Hypocotyl Elongation. Plant Physiol. 48:513-516. 33. Palmer, J. M. 1970. The Influence of Microbial Contamination of Fresh and Washed Beetroot Disks on their Capacity to Absorb Phosphate. Planta 93:'*8-52. 3^- , and B. C. Loughman. I963. Some Factors Affecting the Rate of Absorption and Utilization of Ions by Stem Segments. New Phytol. 63:217-231. 35- , and G. E. Blackman. 1964. Prevention by 2,4- Dichlorophenoxyacet ic Acid of an Increase in the Rate of Phosphate Utilization, Caused by Prolonged Treatment of Segments of Pea Stems in Maleate Buffer. Nature 203:526-527. 36. Pitman, M. G. 1963- The Determination of the Salt Relations of the Cytoplasmic Phase in Cells of Beet Root Tissue. Aust. J. Biol. Sci. 16:647-668. 37. . 1967. Conflicting Measurements of Sodium and Potassium Uptake by Barley Roots. Nature 216:1343-1344. 38. . 1971- Uptake and Transport of Ions in Barley Seed- lings. I. Estimation of Chloride Fluxes in Cells of Excised Roots. Aust. J. Biol. Sci. 24:407-421. 39. . 1969. Adaptation of Barley Roots to Low Oxygen Supply and |ts Relation to Potassium and Sodium Uptake. Plant Physiol. 44:1233-1240. 40. , S. M. Mertz, Jr., J. S. Graves, W. S. Pierce, and N. Higinbotham. 1970. Electrical Potential Differences in Cells of Barley Roots and their Relation to Ion Uptake. Plant Physiol. 47:76-80. 41. Rains, D. W. 1969- Sodium and Potassium Absorption by Bean Stem Tissue. Plant Physiol. 44:547-554. 72 m. , and R. A. Floyd. 1970. Influence of Calcium on Sodium and Potassium Absorption by Fresh and aged Bean Stem Slices. Plant Physiol. '46:93-98. h3. Sacher, J. A. I967. Dual Effect of Auxin: Inhibition of Uptake and Stimulation of RNA and Protein Synthesis: Assessment of Synthesis. Z. Pf lanzenphys iol . 56:4lO-'426. l^k. Sigiura, M. I963. Inhibitory Effect of Kinetin on Respiration of Tobacco Leaf Disks. Botan. Mag. 76:359-362. kS- Smith, R. C. 1970. Time Course of Exudation from Excised Corn Root Segments of Different Stages of Development. Plant Physiol. A5:571-575. 1,6. , B. H. St. John, and R. T. Parrondo. 1973- Influence of Mannitol on Absorption and Retention of Rubidium by Excised Corn Roots. Am. J. Bot . (in press). i»7. Steward, F. C, and J. F. Sutcliff. 1959. Plants in Relation to Inorganic Salts. In: F. C. Steward, ed.. Plant Physiology, a • Treatise, Vol. II. Academic Press, New York, pp 253-'<79- A8. Tanada, T. 1962. Localization and Mechanism of Calcium Stimula- tion of Rubidium Absorption in the Mung Bean Root. Am. J. Bot. i»9: 1068-1072. kS. Yu, G. H., and P. J. Kramer. I967. Radial Salt Transport in Corn Roots. Plant Physiol. '42:985-990. 50, , . 1969. Radial Transport of Ions in Roots Plant Physi oT7T5TT095- H 00 . BIOGRAPHICAL SKETCH Rolando T. Parrondo was born in Camaguey, Cuba, on December 23, 1937. He attended high school at Coleglo Plnson, in Cuba, in I96I, he came to the United States to attend Andrew Junior College, in Cuthbert, Georgia. In 1962, he attended Auburn University, in Auburn, Alabama, and later transferred to George Peabody College for Teachers, in Nashville, Tennessee, where he received a B.S. degree in Biology. Before returning to the University for graduate studies, he taught biology and chemistry at Englewood High School in Jacksonville, Florida. In 1968, he entered graduate school at the University of Florida. In 1969, he received an M.S. degree in Botany. From 1969, to the present time, he has pursued work toward the degree of Doctor of Philosophy. While a graduate student he was a teaching assistant in general botany, general biology, and plant physiology. He is a member of the Botanical Society of America, and the American Society of Plant Physiologists. Rolando T. Parrondo is married to the former Cynthia Cora Melton. 73 1 certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is full' adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. RVchard C. Smith, Chairman Associate Professor of Botany I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Arf^lY // ^-ffS Robert H. Biggs // Professor of Fruit Crops I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Danfei G. Griffin, I I 1^^ Asiociate Professor i^ Botany I certify that ! have read this study and that in my opinion it v,v... forms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. con David Professor of Botany This dissertation was submitted to the Dean of the College of Agricul ture and to the Graduate Council, and was accepted as partial fulfill ment of the requirements for the degree of Doctor of Philosophy. December, 1973 04^ P^ah, College of Agriculture Dean, Graduate School ^'> 0