Planta 139, 25
Planta
28 (1978)
9 by Springer-Verlag 1978
An Evaluation of the Miineh Hypothesis for Phloem Transport in Soybean Donald B. Fisher Department of Botany, University of Georgia, Athens, GA 30602, USA
Abstract. An evaluation was made of the extent which a Miinch-type pressure flow mechanism (i.e., osmotically-generated pressure flow) might contribute to phloem transport in soybean. Estimates of sucrose concentrations in source (leaf) and sink (root) sieve tubes were obtained by a negativestaining procedure. Water potential measurements of the leaf and of the nutrient solution allowed calculation of the turgot pressures in source and sink sieve tubes. The turgot difference between source and sink sieve tubes was compared to that required to drive translocation at the observed velocity between the source and sink, as measured by [14C] photosynthate movement. Sieve-tube conductivity was calculated from the sieve-tube dimensions, assuming an essentially unobstructed pathway. In three experiments, the sucrose concentration was consistently higher in source sieve tubes (an average of 11.5 %) than in sink sieve tubes (an average of 5.3 %). The ratio of these values (2.3: 1) agreed reasonably well with an earlier ratio for source/sink sieve tube concentrations of 1.8:1, obtained by quantitative microautoradiography. The resulting calculated turgot difference (an average of 4.1 bars) was adequate to drive a pressure flow mechanism at the observed translocation velocities (calculated to require a turgot difference of 1.2 to 4.6 bars). No other force need be presumed to be involved. Key words: Glycine Phloem (phloem) - Turgor.
Sieve tubes -
Translocation
croautoradiography to estimate sucrose concentrations in source and sink sieve-tube elements. Their approach was to label the entire shoot for several hours with 1~CO 2 to bring all of the sucrose in the translocation stream to the same (known) specific activity. Sucrose concentrations in leaf and root sieve elements were obtained from the number of disintegrations per unit volume in the sieve-element lumen. Sieve-tube turgot was calculated from these measurements and the water potentials in the leaf and root. The turgor difference between leaf and root was compared to that required to drive translocation at the observed velocity (measured from the rate of [14CJphotosynthate transport) against the resistance to flow in the sieve tubes, shown by Fisher (1975) to be essentially free of obstruction to flow. The ratio of sucrose concentration in the source to that in the sink was found to be about 1.8:1. However, the absolute concentrations (an average of 4.6 % in the source and 2.4 % in the sink) would have been insufficient to maintain positive turgor, and the pressure differences would not have accounted for movement at the observed velocities. This discrepancy could be reasonably attributed to the partial loss of [14C]sucrose during exposure of the sections to water, but this loss could not be quantified. The present report describes a similar approach to the problem, but relies on a different procedure, negative staining of the sieve element contents (Fisher, 1978), to estimate sucrose concentration in the sieve tubes. The results provide clear support for the Miinch hypothesis.
Introduction
Material and Methods
In recent experiments to evaluate the extent to which pressure flow, as envisaged by Miinch (1930), might contribute to phloem transport in soybean, Housley and Fisher (1977) attempted to use quantitative mi-
Growth of Plants
* This work was presented in part at a joint U.S.-Australian Conference on Transport and Transfer Processes in Plants, Canberra, Australia, December 1550, 1975; see Fisher (1976)
Soybean plants (Glycine max (L.) Merr., cv. "Bragg") were grown in Hoagland solution for 5 8 weeks in a growth cabinet. A mixture of fluorescent and incandescent lights provided a photosynthetically active radiation intensity of about 100gE m - 2 s - 1 (about 5,0001x). The plants were grown on 14-h photoperiods at 25~ and a night temperature of 21~ After the first trifoliate leaf was mature, the two simple leaves and cotyledons were removed. The plants were
26
D.B. Fisher: MiJnch Hypothesis for Phloem Transport in Soybean
trimmed once a week so that the two youngest mature trifoliate leaves were the only mature leaves on the plant.
sieve-plate pores are essentially free from obstruction (Fisher, 1975). The pressure difference required to cause pressure flow at the observed velocity in a given experiment was calculated from the total path resistance (1/conductivity), calculated by summing the resistance offered by the petiole, stem and root pathways.
Ttssue Sampling and Measurement of the Translocation Velocity
Several hours before the experiment, the plant was transferred to a fume hood and the lowermost trifoliate leaf was sealed into a plexiglass chamber, with the chamber top removed. A light intensity of 300 gE m- 2s-1 wa supplied by two "Cool Beam" 150-W flood lamps (General Electric Co.), filtered through 5 cm of water. The experiment was started by closing the chamber and labeling the leaf with 250 ~tCi of 14COz for 5 min, after which the chamber top was removed in the draft of the hood. Progress of [14C]photosynthate into the root system was roughly monitored by removing one or two of the shorter roots at appropriate times and checking for the presence of 1~C. When it was estimated that the front was nearing the region in the long root to be used as the sink sample, the experiment was terminated. A leaf punch was taken from each of the two lateral leaflets of the source leaf for water potential measurements. The petiolule of the center leaflet was frozen by dousing it with a mixture of isopentanemethylcyclohexane at -170~ followed with powdered dry ice. Immediately afterwards, a segment of one of the longest roots, about 5 cm from the tip, was also quick-frozen. The remaining length of the root was cut into 2-cm segments; these were dried and assayed for 14C to provide a more accurate velocity measurement. Measurement of Sucrose Concentrations in Sieve-tube Elements
Frozen pieces of the petiolule and of the sink root were freezesubstituted in acetone at - 6 5 ~ to retain their water-soluble organic compounds (Fisher and Housley, 1972). Measurements of sucrose concentrations in the petiolule (source) and root (sink) sieve elements were made by the negative-staining procedure described by Fisher (1978). In brief, this consists of embedding the freeze-substituted tissue in Epon containing 6 ~o (w/v) Sudan B, sectioning at 1.5gm, and making microspectrophotometric measurements of the decrease in staining intensity caused by the presence of sucrose in the sieve elements. As a control, to verify that the negative staining in the sieve elements was caused by water-soluble material, pieces of petiolule were freeze-substituted in methanol, which removes water-soluble organic compounds (Fisher, 1976).
Results F i g u r e s 1 a n d 2 i l l u s t r a t e the a p p e a r a n c e o f s e c t i o n s f r o m n e g a t i v e l y - s t a i n e d p e t i o l u l e a n d r o o t tissues, r e s p e c t i v e l y . N e g a t i v e s t a i n i n g was m o s t p r o n o u n c e d in t h e sieve t u b e s a n d c o m p a n i o n cells. I n c o n t r a s t to o b s e r v a t i o n s m a d e o n b e a n p h l o e m (Fisher, 1978), n e g a t i v e s t a i n i n g was m o r e p r o n o u n c e d in t h e c o m p a n i o n cells t h a n in t h e sieve tubes. S i e v e t u b e s in r o o t p h l o e m w e r e difficult to i d e n t i f y p o s i t i v e l y in a n y g i v e n section. H o w e v e r , m o r e t h a n 50 serial sections were made of each root, allowing positive i d e n t i f i c a t i o n of 8 - 1 2 sieve t u b e s ; t h e o p t i c a l - d e n s i t y m e a s u r e m e n t s w e r e t a k e n f r o m these. T h e s e p r o v e d to be the cells s h o w i n g t h e g r e a t e s t d e g r e e of n e g a t i v e staining. T h a t t h e d e c r e a s e d s t a i n i n g in sieve t u b e s a n d c o m p a n i o n cells was b a s e d u p o n t h e p r e s e n c e of w a t e r - s o l u b l e c o m p o u n d s c a n be seen in t h e s e c t i o n f r o m tissue w h i c h was f r e e z e - s u b s t i t u t e d i n m e t h a n o l (Fig. 3). In t h e l a t t e r tissue, n o n e g a t i v e s t a i n i n g was e v i d e n t in t h e sieve e l e m e n t s ; this was v e r i f i e d b y microspectrophotometric measurements. However, m o s t p a r e n c h y m a cells still s h o w e d s o m e n e g a t i v e staining. T h i s is p o s s i b l y c a u s e d b y t h e p r e s e n c e of r e l a t i v e l y m o r e i n o r g a n i c s o l u t e s in t h o s e cells; t h e i r s o l u b i l i t y d u r i n g f r e e z e - s u b s t i t u t i o n in m e t h a n o l has not been investigated.
Water-potential Measurements
Leaf water-potential measurements were made with thermocouple psychrometers, using the isopiestic technique desribed by Boyer and Knipling (1965). The estimated accuracy of the measurements was _+0.3 0.5bars, depending on the thermocouple. The water potential of the nutrient solution was measured by vapor pressure osmometry, with an accuracy o[ _+0.04bars (Housley and Fisher, 1977). Sieve-tube Conductivities and Calculation of the Pressure Difference Required for Flow
Values for sieve-tube conductivities (i.e., cm min-1/barcm -1) in the petiole, stem and root were taken to be the same as previously reported for soybean plants grown under identical conditions (Housley and Fisher, 1977). The conductivities were calculated from the Hagen-Poiseuille equation, with the assumption that the
Fig. 1. A cross-section, 1.5 gm thick, through vascular tissue in a soybean petiolule which was freeze-substituted in acetone and embedded in Epon containing 6 ~o Sudan B. Negative staining is most pronounced in the sieve elements and companion cells, with the smaller companion cells staining more lightly than the sieve elements. Ca. 16 sieve element-companion cell pairs are visible. Xylem vessels appear in the left part of the micrograph, x 440
D.B. Fisher: Mtinch Hypothesis for Phloem Transport in Soybean
27 Table 1. Path lengths, velocities and the required turgor differences between the source and sink to drive pressure flow at the observed velocities
A$p (bars) required with Experiment Path length Velocity (cm) (cm m i n - 1) the sieve plate pores
A B C
70 123 160
0.8 0.9 0.8
100~open
70~open"
1.2 1.9 2.0
2.7 4.2 4.6
i.e., pore diameter=0.7 x the measured diameter
Table 2. Osmotic, water and turgor potentials in sieve tubes Fig. 2. A cross-section, 1.5 ~tm thick, through vascular tissue in a soybean root which was freeze-substituted in acetone and embedded in Epon containing 6 ~ Sudan B. Positive identification of some of the cells as sieve elements was made by examination of serial sections. They are located in the band of smaller cells running along the upper part of the micrograph and exhibited the strongest negative staining of all cells in the stele. Xylem vessels appear in the lower left and right of the micrograph, x 440
Experiment
Location
Sucrose concn.
Os (bars)
~',,. (bars)
@ A@ (bars) (bars)
(%) A
Petiolule Root
11.5_+2.9 4.4+_0.9
-
9.7 3.3
-4.3 a -2.0 b
5.4 1.3
4.1
B
Petiolule Root
10.5+_0.9 4.5_+1.1
-
8.7 3.4
-3.4 ~ -2.0 b
5.3 1.4
3.9
C
Petiolule Root
12.5+ 1.4 6.3+_0.4
- 10.7 - 4.9
-3.4" -2.0 b
7.3 2.9
4.4
Measured in the leaf by thermocouple psychrometry b Estimated; nutrient ~,~ was - 0 . 6 b a r
Fig. 3. A cross-section, 1.5 g m thick, of negatively-stained soybean petiolule which was freeze-substituted in methanol to remove water-soluble organic compounds. Nine sieve element-companion cell pairs are visible in the right region of the micrograph, with xylem vessels in the lower left. Note the absence of negative staining in the sieve tubes and companion cells, x 440
Three separate experiments were run, using a range of total path lengths between source and sink. Table 1 gives the path lengths, velocities and turgor gradients necessary to drive pressure-generated flow at the observed velocities along the path. Because there are several sources of error in the calculated values for conductivities (Housley and Fisher, 1977), arising particularly from the sensitivity of the calculation to sieve-plate pore size, two values are given for the required pressure drop. The relative values for
the required pressure drop in different experiments are not proportional to the path lengths. This arises from the fact that the stem, the length of which accounted for most of the difference in path lengths, has a substantially higher conductivity than the petiole or root. Values for the sucrose concentrations and the osmotic, turgot and water potentials in root and shoot sieve elements are given in Table 2. The values given for sucrose concentrations and osmotic potentials assume that all of the solute in the sieve tubes was in the form of sucrose. Each concentration measurement comes from 70 to 130 optical-density measurements on 8 to 16 sieve tubes. There was little variation between experiments in the source or sink sucrose concentrations or, consequently, in the source or sink solute potentials in the sieve tubes. Leaf water potentials were also quite similar. Water potentials in the root could not be measured directly but, on the basis of the leaf and nutrient-solution water potentials (the latter was - 0 . 6 b a r s ) , were estimated at - 2 b a r s (see Discussion). Given these values of osmotic and water potentials, the calculated sieve-tube turgor was positive in all cases. A substantial turgor difference apparently existed between source and sink. The calculated turgor difference was comparable to that required to drive a simple pressure-flow mechanism at the observed velocities (Table 1).
28
D.B. Fisher: Miinch Hypothesis for Phloem Transport in Soybean
Discussion
A second possibility for error in the turgor calculations arises from the fact that the data do not allow direct calculation of sieve-tube turgor in the root 9because the water potential could not be measured within the stele. Hence, the size of the calculated turgot difference depends on what assumptions are made about the stelar water potential. A value of - 2 bars was chosen on the basis of the high resistance to water flow in soybean roots (Boyer, 1971) and the measured values for water potential in the hypocotyl xylem of solution-grown soybean plants (Michel, 1977). It seems quite unlikely that the true value would be more than +1 bar removed from the assumed figure. This would not alter the basic conclusion to be drawn from the data: the Mfinch hypothesis of osmotically-generated pressure flow seems adequate to account for phloem transport in soybean.
To verify the Mtinch hypothesis of phloem transport, it is necessary to demonstrate that there is a sufficient turgor gradient along the sieve tubes to cause movement at the observed velocity against the resistance to flow offered by the sieve-tubes. Until our previous effort, which employed quantitative microautoradiography to estimate sucrose concentrations (Housley and Fisher, 1977), no attempt had been made to obtain measurements of resistance, velocity and turgor gradient from the same plant. Our experiments did not yield reliable values for absolute sucrose concentrations (a fraction of the sucrose was lost upon exposure of the sections to water), but they showed that the ratio of sucrose concentrations in source and sink sieve tubes was 1.8: 1. This compares favorably to the ratio of 2.3:1 in the present experiments. The absolute concentrations obtained from the negative staining procedure are more realistic than those from microautoradiography. They are comparable to sugar concentrations in many phloem exudates, including those from herbaceous species (Fife et al., 1962; see review by Pate, 1975). The concentrations would have been sufficiently high to maintai/a positive turgor in the sieve tubes. Most importantly, the present measurements strongly indicate that the turgor differences between leaf and root sieve tubes were sufficienly great to drive a Mtinch-type pressure-flow mechanism of phloem transport. The calculated pressure differences (Table 2) exceeded or matched the required pressure differences (Table 1), even when fairly restrictive assumptions were made for the sieve-plate pore pathway. It should be recognized, however, that the values given for sucrose concentrations and osmotic potentials were calculated by assuming that no solutes other than sucrose were present in the sieve tubes. The probable consequences of this assumption were discussed in another paper (Fisher, 1978): since most of the other solutes are probably ionizable, this would have the effect of somewhat overestimating the sucrose concentration, but underestimating the osmotic potential. If the relative contributions of various compounds to the osmotic potentials in the source and sink were similar, this would make the actual osmotic-potenial difference between source and sink somewhat greater than the values given in Table 2. Therefore, the direction of the error incurred by the assumption is most likely in the direction of an even greater turgor difference between source and sink than the calculated values.
Supported by National Science Foundation Grants GB 33905 and BMS 7509337.
References Boyer, J.S.: Resistances to water transport in soybean, bean and sunflower. Crop Sci. 11, 403-407 (1971) Boyer, J.S., Knipling, E.B.: Isopiestic technique for measuring leaf water potentials with a thermocouple psychrometer. Proc. Natl. Acad. Sci. USA 54, 1044-1051 (1965) Fife, J.M., Price, C., Fife, D.C.: Some properties of phloem exudate collected from root of sugar beet. Plant Physiol. 37, 791-792 (1962) Fisher, D.B.: Structure of functional soybean sieve elements. Plant Physiol. 56, 555-569 (1975) Fisher, D.B.: Histochemical approaches to water-soluble compounds and their application to problems in translocation. In: Transport and Transfer Processes in Plants, pp. 237-246, Wardlaw, I.F., Passioura, J.B., eds. New York: Academic Press 1976 Fisher, D.B.: The estimation of sugar concentrations in individual sieve tube elements by negative staining. Planta 139, 19-24 (1978) Fisher, D.B., Housley, T.L.: The retention of water-soluble compounds during freeze-substitution and microautoradiography. Plant Physiol. 49, 166-171 (1972) Housley, T.L., Fisher, D.B.: Estimation of osmotic gradients in soybean sieve tubes. Qualified support for the Miinch hypothesis. Plant Physiol. 59, 701 706 (1977) Michel, B.E.: A miniature stem thermocouple hygrometer. Plant Physiol. 60, 645-647 (1977) Miinch, E.: Die Stoffbewegungen in der Pflanze. Jena: Fischer 1930 Pate, J.S.: Exchange of solutes between phloem and xylem and circulation in the whole plant. In: Transport in Plants. I. Phloem Transport, pp. 451~473, Zimmermann, M.H., Milburn, J.A., eds. (Encycl. Plant Physiol., N_S., Risson, A., Zimmermann, M.H., eds., vol. 1.) Berlin-Heidelberg-New York: Springer 1975 Received 19 September; accepted 18 November 1977
Planta
28 (1978)
9 by Springer-Verlag 1978
An Evaluation of the Miineh Hypothesis for Phloem Transport in Soybean Donald B. Fisher Department of Botany, University of Georgia, Athens, GA 30602, USA
Abstract. An evaluation was made of the extent which a Miinch-type pressure flow mechanism (i.e., osmotically-generated pressure flow) might contribute to phloem transport in soybean. Estimates of sucrose concentrations in source (leaf) and sink (root) sieve tubes were obtained by a negativestaining procedure. Water potential measurements of the leaf and of the nutrient solution allowed calculation of the turgot pressures in source and sink sieve tubes. The turgot difference between source and sink sieve tubes was compared to that required to drive translocation at the observed velocity between the source and sink, as measured by [14C] photosynthate movement. Sieve-tube conductivity was calculated from the sieve-tube dimensions, assuming an essentially unobstructed pathway. In three experiments, the sucrose concentration was consistently higher in source sieve tubes (an average of 11.5 %) than in sink sieve tubes (an average of 5.3 %). The ratio of these values (2.3: 1) agreed reasonably well with an earlier ratio for source/sink sieve tube concentrations of 1.8:1, obtained by quantitative microautoradiography. The resulting calculated turgot difference (an average of 4.1 bars) was adequate to drive a pressure flow mechanism at the observed translocation velocities (calculated to require a turgot difference of 1.2 to 4.6 bars). No other force need be presumed to be involved. Key words: Glycine Phloem (phloem) - Turgor.
Sieve tubes -
Translocation
croautoradiography to estimate sucrose concentrations in source and sink sieve-tube elements. Their approach was to label the entire shoot for several hours with 1~CO 2 to bring all of the sucrose in the translocation stream to the same (known) specific activity. Sucrose concentrations in leaf and root sieve elements were obtained from the number of disintegrations per unit volume in the sieve-element lumen. Sieve-tube turgot was calculated from these measurements and the water potentials in the leaf and root. The turgor difference between leaf and root was compared to that required to drive translocation at the observed velocity (measured from the rate of [14CJphotosynthate transport) against the resistance to flow in the sieve tubes, shown by Fisher (1975) to be essentially free of obstruction to flow. The ratio of sucrose concentration in the source to that in the sink was found to be about 1.8:1. However, the absolute concentrations (an average of 4.6 % in the source and 2.4 % in the sink) would have been insufficient to maintain positive turgor, and the pressure differences would not have accounted for movement at the observed velocities. This discrepancy could be reasonably attributed to the partial loss of [14C]sucrose during exposure of the sections to water, but this loss could not be quantified. The present report describes a similar approach to the problem, but relies on a different procedure, negative staining of the sieve element contents (Fisher, 1978), to estimate sucrose concentration in the sieve tubes. The results provide clear support for the Miinch hypothesis.
Introduction
Material and Methods
In recent experiments to evaluate the extent to which pressure flow, as envisaged by Miinch (1930), might contribute to phloem transport in soybean, Housley and Fisher (1977) attempted to use quantitative mi-
Growth of Plants
* This work was presented in part at a joint U.S.-Australian Conference on Transport and Transfer Processes in Plants, Canberra, Australia, December 1550, 1975; see Fisher (1976)
Soybean plants (Glycine max (L.) Merr., cv. "Bragg") were grown in Hoagland solution for 5 8 weeks in a growth cabinet. A mixture of fluorescent and incandescent lights provided a photosynthetically active radiation intensity of about 100gE m - 2 s - 1 (about 5,0001x). The plants were grown on 14-h photoperiods at 25~ and a night temperature of 21~ After the first trifoliate leaf was mature, the two simple leaves and cotyledons were removed. The plants were
26
D.B. Fisher: MiJnch Hypothesis for Phloem Transport in Soybean
trimmed once a week so that the two youngest mature trifoliate leaves were the only mature leaves on the plant.
sieve-plate pores are essentially free from obstruction (Fisher, 1975). The pressure difference required to cause pressure flow at the observed velocity in a given experiment was calculated from the total path resistance (1/conductivity), calculated by summing the resistance offered by the petiole, stem and root pathways.
Ttssue Sampling and Measurement of the Translocation Velocity
Several hours before the experiment, the plant was transferred to a fume hood and the lowermost trifoliate leaf was sealed into a plexiglass chamber, with the chamber top removed. A light intensity of 300 gE m- 2s-1 wa supplied by two "Cool Beam" 150-W flood lamps (General Electric Co.), filtered through 5 cm of water. The experiment was started by closing the chamber and labeling the leaf with 250 ~tCi of 14COz for 5 min, after which the chamber top was removed in the draft of the hood. Progress of [14C]photosynthate into the root system was roughly monitored by removing one or two of the shorter roots at appropriate times and checking for the presence of 1~C. When it was estimated that the front was nearing the region in the long root to be used as the sink sample, the experiment was terminated. A leaf punch was taken from each of the two lateral leaflets of the source leaf for water potential measurements. The petiolule of the center leaflet was frozen by dousing it with a mixture of isopentanemethylcyclohexane at -170~ followed with powdered dry ice. Immediately afterwards, a segment of one of the longest roots, about 5 cm from the tip, was also quick-frozen. The remaining length of the root was cut into 2-cm segments; these were dried and assayed for 14C to provide a more accurate velocity measurement. Measurement of Sucrose Concentrations in Sieve-tube Elements
Frozen pieces of the petiolule and of the sink root were freezesubstituted in acetone at - 6 5 ~ to retain their water-soluble organic compounds (Fisher and Housley, 1972). Measurements of sucrose concentrations in the petiolule (source) and root (sink) sieve elements were made by the negative-staining procedure described by Fisher (1978). In brief, this consists of embedding the freeze-substituted tissue in Epon containing 6 ~o (w/v) Sudan B, sectioning at 1.5gm, and making microspectrophotometric measurements of the decrease in staining intensity caused by the presence of sucrose in the sieve elements. As a control, to verify that the negative staining in the sieve elements was caused by water-soluble material, pieces of petiolule were freeze-substituted in methanol, which removes water-soluble organic compounds (Fisher, 1976).
Results F i g u r e s 1 a n d 2 i l l u s t r a t e the a p p e a r a n c e o f s e c t i o n s f r o m n e g a t i v e l y - s t a i n e d p e t i o l u l e a n d r o o t tissues, r e s p e c t i v e l y . N e g a t i v e s t a i n i n g was m o s t p r o n o u n c e d in t h e sieve t u b e s a n d c o m p a n i o n cells. I n c o n t r a s t to o b s e r v a t i o n s m a d e o n b e a n p h l o e m (Fisher, 1978), n e g a t i v e s t a i n i n g was m o r e p r o n o u n c e d in t h e c o m p a n i o n cells t h a n in t h e sieve tubes. S i e v e t u b e s in r o o t p h l o e m w e r e difficult to i d e n t i f y p o s i t i v e l y in a n y g i v e n section. H o w e v e r , m o r e t h a n 50 serial sections were made of each root, allowing positive i d e n t i f i c a t i o n of 8 - 1 2 sieve t u b e s ; t h e o p t i c a l - d e n s i t y m e a s u r e m e n t s w e r e t a k e n f r o m these. T h e s e p r o v e d to be the cells s h o w i n g t h e g r e a t e s t d e g r e e of n e g a t i v e staining. T h a t t h e d e c r e a s e d s t a i n i n g in sieve t u b e s a n d c o m p a n i o n cells was b a s e d u p o n t h e p r e s e n c e of w a t e r - s o l u b l e c o m p o u n d s c a n be seen in t h e s e c t i o n f r o m tissue w h i c h was f r e e z e - s u b s t i t u t e d i n m e t h a n o l (Fig. 3). In t h e l a t t e r tissue, n o n e g a t i v e s t a i n i n g was e v i d e n t in t h e sieve e l e m e n t s ; this was v e r i f i e d b y microspectrophotometric measurements. However, m o s t p a r e n c h y m a cells still s h o w e d s o m e n e g a t i v e staining. T h i s is p o s s i b l y c a u s e d b y t h e p r e s e n c e of r e l a t i v e l y m o r e i n o r g a n i c s o l u t e s in t h o s e cells; t h e i r s o l u b i l i t y d u r i n g f r e e z e - s u b s t i t u t i o n in m e t h a n o l has not been investigated.
Water-potential Measurements
Leaf water-potential measurements were made with thermocouple psychrometers, using the isopiestic technique desribed by Boyer and Knipling (1965). The estimated accuracy of the measurements was _+0.3 0.5bars, depending on the thermocouple. The water potential of the nutrient solution was measured by vapor pressure osmometry, with an accuracy o[ _+0.04bars (Housley and Fisher, 1977). Sieve-tube Conductivities and Calculation of the Pressure Difference Required for Flow
Values for sieve-tube conductivities (i.e., cm min-1/barcm -1) in the petiole, stem and root were taken to be the same as previously reported for soybean plants grown under identical conditions (Housley and Fisher, 1977). The conductivities were calculated from the Hagen-Poiseuille equation, with the assumption that the
Fig. 1. A cross-section, 1.5 gm thick, through vascular tissue in a soybean petiolule which was freeze-substituted in acetone and embedded in Epon containing 6 ~o Sudan B. Negative staining is most pronounced in the sieve elements and companion cells, with the smaller companion cells staining more lightly than the sieve elements. Ca. 16 sieve element-companion cell pairs are visible. Xylem vessels appear in the left part of the micrograph, x 440
D.B. Fisher: Mtinch Hypothesis for Phloem Transport in Soybean
27 Table 1. Path lengths, velocities and the required turgor differences between the source and sink to drive pressure flow at the observed velocities
A$p (bars) required with Experiment Path length Velocity (cm) (cm m i n - 1) the sieve plate pores
A B C
70 123 160
0.8 0.9 0.8
100~open
70~open"
1.2 1.9 2.0
2.7 4.2 4.6
i.e., pore diameter=0.7 x the measured diameter
Table 2. Osmotic, water and turgor potentials in sieve tubes Fig. 2. A cross-section, 1.5 ~tm thick, through vascular tissue in a soybean root which was freeze-substituted in acetone and embedded in Epon containing 6 ~ Sudan B. Positive identification of some of the cells as sieve elements was made by examination of serial sections. They are located in the band of smaller cells running along the upper part of the micrograph and exhibited the strongest negative staining of all cells in the stele. Xylem vessels appear in the lower left and right of the micrograph, x 440
Experiment
Location
Sucrose concn.
Os (bars)
~',,. (bars)
@ A@ (bars) (bars)
(%) A
Petiolule Root
11.5_+2.9 4.4+_0.9
-
9.7 3.3
-4.3 a -2.0 b
5.4 1.3
4.1
B
Petiolule Root
10.5+_0.9 4.5_+1.1
-
8.7 3.4
-3.4 ~ -2.0 b
5.3 1.4
3.9
C
Petiolule Root
12.5+ 1.4 6.3+_0.4
- 10.7 - 4.9
-3.4" -2.0 b
7.3 2.9
4.4
Measured in the leaf by thermocouple psychrometry b Estimated; nutrient ~,~ was - 0 . 6 b a r
Fig. 3. A cross-section, 1.5 g m thick, of negatively-stained soybean petiolule which was freeze-substituted in methanol to remove water-soluble organic compounds. Nine sieve element-companion cell pairs are visible in the right region of the micrograph, with xylem vessels in the lower left. Note the absence of negative staining in the sieve tubes and companion cells, x 440
Three separate experiments were run, using a range of total path lengths between source and sink. Table 1 gives the path lengths, velocities and turgor gradients necessary to drive pressure-generated flow at the observed velocities along the path. Because there are several sources of error in the calculated values for conductivities (Housley and Fisher, 1977), arising particularly from the sensitivity of the calculation to sieve-plate pore size, two values are given for the required pressure drop. The relative values for
the required pressure drop in different experiments are not proportional to the path lengths. This arises from the fact that the stem, the length of which accounted for most of the difference in path lengths, has a substantially higher conductivity than the petiole or root. Values for the sucrose concentrations and the osmotic, turgot and water potentials in root and shoot sieve elements are given in Table 2. The values given for sucrose concentrations and osmotic potentials assume that all of the solute in the sieve tubes was in the form of sucrose. Each concentration measurement comes from 70 to 130 optical-density measurements on 8 to 16 sieve tubes. There was little variation between experiments in the source or sink sucrose concentrations or, consequently, in the source or sink solute potentials in the sieve tubes. Leaf water potentials were also quite similar. Water potentials in the root could not be measured directly but, on the basis of the leaf and nutrient-solution water potentials (the latter was - 0 . 6 b a r s ) , were estimated at - 2 b a r s (see Discussion). Given these values of osmotic and water potentials, the calculated sieve-tube turgor was positive in all cases. A substantial turgor difference apparently existed between source and sink. The calculated turgor difference was comparable to that required to drive a simple pressure-flow mechanism at the observed velocities (Table 1).
28
D.B. Fisher: Miinch Hypothesis for Phloem Transport in Soybean
Discussion
A second possibility for error in the turgor calculations arises from the fact that the data do not allow direct calculation of sieve-tube turgor in the root 9because the water potential could not be measured within the stele. Hence, the size of the calculated turgot difference depends on what assumptions are made about the stelar water potential. A value of - 2 bars was chosen on the basis of the high resistance to water flow in soybean roots (Boyer, 1971) and the measured values for water potential in the hypocotyl xylem of solution-grown soybean plants (Michel, 1977). It seems quite unlikely that the true value would be more than +1 bar removed from the assumed figure. This would not alter the basic conclusion to be drawn from the data: the Mfinch hypothesis of osmotically-generated pressure flow seems adequate to account for phloem transport in soybean.
To verify the Mtinch hypothesis of phloem transport, it is necessary to demonstrate that there is a sufficient turgor gradient along the sieve tubes to cause movement at the observed velocity against the resistance to flow offered by the sieve-tubes. Until our previous effort, which employed quantitative microautoradiography to estimate sucrose concentrations (Housley and Fisher, 1977), no attempt had been made to obtain measurements of resistance, velocity and turgor gradient from the same plant. Our experiments did not yield reliable values for absolute sucrose concentrations (a fraction of the sucrose was lost upon exposure of the sections to water), but they showed that the ratio of sucrose concentrations in source and sink sieve tubes was 1.8: 1. This compares favorably to the ratio of 2.3:1 in the present experiments. The absolute concentrations obtained from the negative staining procedure are more realistic than those from microautoradiography. They are comparable to sugar concentrations in many phloem exudates, including those from herbaceous species (Fife et al., 1962; see review by Pate, 1975). The concentrations would have been sufficiently high to maintai/a positive turgor in the sieve tubes. Most importantly, the present measurements strongly indicate that the turgor differences between leaf and root sieve tubes were sufficienly great to drive a Mtinch-type pressure-flow mechanism of phloem transport. The calculated pressure differences (Table 2) exceeded or matched the required pressure differences (Table 1), even when fairly restrictive assumptions were made for the sieve-plate pore pathway. It should be recognized, however, that the values given for sucrose concentrations and osmotic potentials were calculated by assuming that no solutes other than sucrose were present in the sieve tubes. The probable consequences of this assumption were discussed in another paper (Fisher, 1978): since most of the other solutes are probably ionizable, this would have the effect of somewhat overestimating the sucrose concentration, but underestimating the osmotic potential. If the relative contributions of various compounds to the osmotic potentials in the source and sink were similar, this would make the actual osmotic-potenial difference between source and sink somewhat greater than the values given in Table 2. Therefore, the direction of the error incurred by the assumption is most likely in the direction of an even greater turgor difference between source and sink than the calculated values.
Supported by National Science Foundation Grants GB 33905 and BMS 7509337.
References Boyer, J.S.: Resistances to water transport in soybean, bean and sunflower. Crop Sci. 11, 403-407 (1971) Boyer, J.S., Knipling, E.B.: Isopiestic technique for measuring leaf water potentials with a thermocouple psychrometer. Proc. Natl. Acad. Sci. USA 54, 1044-1051 (1965) Fife, J.M., Price, C., Fife, D.C.: Some properties of phloem exudate collected from root of sugar beet. Plant Physiol. 37, 791-792 (1962) Fisher, D.B.: Structure of functional soybean sieve elements. Plant Physiol. 56, 555-569 (1975) Fisher, D.B.: Histochemical approaches to water-soluble compounds and their application to problems in translocation. In: Transport and Transfer Processes in Plants, pp. 237-246, Wardlaw, I.F., Passioura, J.B., eds. New York: Academic Press 1976 Fisher, D.B.: The estimation of sugar concentrations in individual sieve tube elements by negative staining. Planta 139, 19-24 (1978) Fisher, D.B., Housley, T.L.: The retention of water-soluble compounds during freeze-substitution and microautoradiography. Plant Physiol. 49, 166-171 (1972) Housley, T.L., Fisher, D.B.: Estimation of osmotic gradients in soybean sieve tubes. Qualified support for the Miinch hypothesis. Plant Physiol. 59, 701 706 (1977) Michel, B.E.: A miniature stem thermocouple hygrometer. Plant Physiol. 60, 645-647 (1977) Miinch, E.: Die Stoffbewegungen in der Pflanze. Jena: Fischer 1930 Pate, J.S.: Exchange of solutes between phloem and xylem and circulation in the whole plant. In: Transport in Plants. I. Phloem Transport, pp. 451~473, Zimmermann, M.H., Milburn, J.A., eds. (Encycl. Plant Physiol., N_S., Risson, A., Zimmermann, M.H., eds., vol. 1.) Berlin-Heidelberg-New York: Springer 1975 Received 19 September; accepted 18 November 1977
