Planta (1989)177 : 327 335
P l a n t a 9 Springer-Verlag 1989
Sucrose transport into the phloem of Ricinus communis L. seedlings as measured by the analysis of sieve-tube sap Jose Kallarackal*, Gabriele Orlich, Christian Schobcrt, and Ewald Komor** Pflanzenphysiologie, Universit/it Bayreuth, Postfach 1012 51, D-8580 Bayreuth, Federal Republic of Germany
Abstract. Careful cutting of the hypocotyl of R i c i nus c o m m u n i s L. seedlings led to the exudation of pure sieve-tube sap for 2-3 h. This offered the possibility of testing the phloem-loading system qualitatively and quantitatively by incubating the cotyledons with different solutes of various concentrations to determine whether or not these solutes were loaded into the sieve tubes. The concentration which was achieved by loading and the time course could also be documented. This study concentrated on the loading of sucrose because it is the major naturally translocated sieve-tube compound. The sucrose concentration of sieve-tube sap was approx. 300 m M when the cotyledons were buried in the endosperm. When the cotyledons were excised from the endosperm and incubated in buffer, the sucrose concentration decreased gradually to 80-100 mM. This sucrose level was maintained for several hours by starch breakdown. Incubation of the excised cotyledons in sucrose caused the sucrose concentration in the sieve tubes to rise from 80 to 400 mM, depending on the sucrose concentration in the medium. Thus the sucrose concentration in the sieve tubes could be manipulated over a wide range. The transfer of labelled sucrose to the sieve-tube sap took I0 min; full isotope equilibration was finally reached after 2 h. An increase of K § in the medium or in the sieve tubes did not change the sucrose concentration in the sievetube sap. Similarly the experimentally induced change of sucrose concentration in the sieve tubes did not affect the K § concentration in the exudate. High concentrations of K § however, strongly re* P r e s e n t a d d r e s s : Plant Physiology Division, Kerala Forest Research Institute, Peechi 680 653, Trichur, Kerala, India ** To whom correspondence should be addressed A b b r e v i a t i o n ." PTS
= 3-hydroxy-5,8,J 0-pyrenetrisulfonate
duced the flow rate of exudation. Similar results were obtained with Na + (data not shown). The minimum translocation speed in the sieve tubes in vivo was calculated from the growth increment of the seedling to be 1.03 m - h 1, a value, which on average was also obtained for the exudation system with the endosperm attached. This comparison of the in-vivo rate of phloem transport and the exudation rate from cut hypocotyls indicates that sink control of phloem transport in the seedlings of that particular age was small, if there was any at all, and that the results from the experimental exudation system were probably not falsified by removal of the sink tissues.
Key words: Exudation (phloem, K + effect) Phloem (translocation speed) Potassium (phloem) - R i c i n u s (phloem transport) - Sink control - Sucrose loading
Introduction The transport of solutes by the phloem is an :important step in the partitioning of assimilates in the plant. However, the mechanism and the specificity of the transport of solutes into the sieve tubes are not known because the complexity of the tissue has not allowed unambiguous kinetic measurements, as is possible for example in homogeneous parenchyma tissues and single cells (reviewed by K o m o r 1982; Kursanov 1984). So far the investigations of phloem loading have been of two types. One approach was the analysis of sieve-tube sap from those (few) plants which either exhibit "phloem bleeding" upon incision of the bark (Hall et al. ]971 ; Smith and Milburn 1980a-c) or which
328
could be infested with aphids, whose stylets could then be cut (Fisher 1981). In this way the composition of sieve-tube sap in plants under natural conditions has been determined. These systems were, however, limited by the difficulty of manipulating, both qualitatively and quantitatively, the composition of the exudate: incubation of the leaf veins was difficult because of the cuticle, and the storage compounds in the leaf "buffered" most of the experimental treatments. For example, darkening the leaves for some hours hardly affected the sucrose concentration in the exuding sap of Ricinus (Smith and Milburn 1980 a). Furthermore, because solutes cannot be applied in definite concentrations to the leaf veins, it could not be decided whether the lack of a particular substance in the sieve-tube sap was because of the unavailability of this substance at the phloem-loading site or because of the incapability of the loading system to take up this substance. The other common type of experiment involved the use of pieces of phloem strands or leaf discs (Sovonick et al. 1974; Daie 1987) which were usually incubated with labelled substances and the uptake of label determined and localized by autoradiography. These studies suffered from the disadvantage that it was difficult to prove that the label, which for instance was found in the veins, was still in the compound which had been supplied and was not in a metabolic product. In addition the labelling of the veins cannot necessarily be considered to be identical to the labelling of the sieve tubes. Therefore, we have tried to elaborate an experimental system, which combines the advantages of the analysis of sieve-tube sap with those of the incubation method, with the aim of investigating the response of the sieve-tube loading system to defined conditions in the leaf. We chose the Ricinus seedling as a model system because the cotyledons, since they do not have a cuticle, readily respond to incubation with solutes (Kriedemann and Beevers 1967a, b). Since the adult Ricinus plant is one of the few, plants, which show phloembleeding after incision, we hoped that cutting or incising the hypocotyl of the seedling might give rise to sieve-tube exudation. With this system it would be possible to follow the loading of substances of interest into the sieve tubes and to reveal which conditions and factors regulate solute transport into the phloem. This report demonstrates that pure sieve-tube sap can be obtained from Ricinus seedlings for several hours after cutting the hypocotyl and that the seedling responds to experimental changes both rapidly and extensively.
J. Kallarackal et al. : Sucrose transport into the sieve-tube of Ricinus
Material and methods Plant material and chemicals'. Seeds of Ricinus communis L. cv. gibsonii were either obtained from greenhouse-grown plants or were purchased from Walz, Stuttgart, F R G . The 14C-labelled compounds were bought from Amersham, Braunschweig (FRG), and N E N - D u p o n t , Dreieich (FRG). 3-Hydroxy-5,8,10pyrenetrisulfonate (PTS) was a gift from Dr. J.J. Jachetta (Cornell University, Ithaca, N.Y.). Growth of seedlings. The seeds were immersed in 0.3% chinosol solution for i0 min and then soaked in water overnight. Initially, they were planted in wet vermiculite the following day, incubated in a humid atmosphere at 27~ in dim light for 5 d, and seedlings of average size then were selected for the experiments. Later, it was found that a better yield of exuding seedlings could be obtained with plants, which were raised axenically in water culture. In that case the seed coat was removed and the naked seed was surface-sterilized in hydrogen peroxide (Schobert and K o m o r 1987). After repeated washings the seeds were germinated for 2 d on agar enriched in organic nutrients to ensure the absence of bacteria. Then the seedlings were transferred to a sterile aerated water culture and were allowed to grow up to the sixth day.
Incubation, cutting and collection of sieve-tube sap. When the seedlings were grown in vermiculite, they were not removed from the substratum, but kept there until the hypocotyls were cut. Water-cultivated seedlings were carefully transferred to a beaker with the roots immersed in 0.5 m M CaC12 at 27 ~ C. The individual steps of the preparation, cutting and sap collection are illustrated in Fig. 1. Depending on the intention of the investigation, the endosperm was carefully removed without bending or squeezing the cotyledons or the hypoeotyl, Then the cotyledons were incubated in buffer solution, e.g. 5 m M potassium phosphate pH 6.3, or in buffer supplemented with sugar, ions or other compounds of interest (the roots still immersed in 0.5 m M CaC12). For exudation the hypocotyl was severed in the hook region approx. 10 m m apart from the cotyledons with a sharp razor blade, using a steady, non-squeezing motion. The time-point of cutting was either at the moment of addition of a particular solute, or after 2 h of incubation with the solute. After cutting, the exudation of sap from the cut end of the upright hypocotyl was observed; in those cases where no exudation occurred, a further slicing of the hypocotyl helped to initiate exudation. The cut hypoeotyl was leaning against the wall of a small beaker or was supported with putty. The sap was usually collected continuously with graded glass capillaries until a 2-gl sample was obtained; the time-point of collection was noted and the 2-I,tl sample was immediately diluted with water to 200 lal and was kept in the cold till analysis. In order to prevent evaporation from the droplet on the exuding hypocotyl stump, the whole incubation was performed in a water bath at 27 ~ C under a Plexiglas hood, which was layed out with wet filter paper. Small openings at the side of the hood allowed insertion of the microcapillaries for exudate collections. Evaporation was experimentally determined by putting a droplet with a defined sucrose concentration onto the stump of a non-exuding seedling. We found that evaporation caused the sucrose concentration to increase by less than 3% within 5 min, and a droplet never remained longer than 5 min on the cut stump. Evaporation from the microcapillaries was negligible (below 2% in 10 rain).
Analytical methods. Sugars, i.e. sucrose, glucose and fructose, were quantified enzymatically with a test set from Boehringer, Mannheim, F R G , or by the anthrone method (Yemm and Wit-
J. Kallarackal et aI. : Sucrose transport into the sieve-tube of Ricinus
Fig. 1 A-E. Illustration of the seedling preparation, cutting and
sieve tube sap collection. A Six days old Ricinus seedlings before (left) and after (right) endosperm removal. B Preincubation of the cotyledons in a defined solution with the roots immersed in 0.5 m M CaC12. The arrow indicates the site of cutting. C Incubation of the cotyledons during exudation. D Sieve tube sap exudation from the hypocotyl stump. E Exudate collection with microcapiilary under the hood of the temperature-controlled water bath
lis 1954). SampIes were chromatographed on thin-layer cellulose gels with ethyl acetate-pyridine-water, 100:35:25 (by vol.) as solvent. The inorganic ions such as K § Na § Ca 2+ and Mg 2+ were determined by atomic absorption (Perkin-Elmer 5000, Norwalk, Conn., USA). The presence of the apoplastic marker 3-hydroxy-5,8,10-pyrenetrisulfonate (PTS) (Dybing and Currier 1961) in the phloem exudate (20-gl samples) was measured in microcuvettes using a fluorescence spectrometer (Perkin-Elmer 650 10 M) at 405-nm excitation and 510-nm emission wavelengths. Radioactivity was measured by scintillation counting in dioxan-naphthalene-diphenyloxazol (1000:100: 5 by vol.). The pH of sieve-tube sap was determined with a flat-bottomed electrode (Ingold, Ziirich, Switzerland) in 10-gl samples.
Results
Establishment o f the exudation system with the endosperm attached to the cotyledons. When the hypocotyl of a 6-d-old Ricinus seedling was cut near the cotyledons just below the hook, an exudation
329
E
-50~
g~ 400-4--
300-
sucrose concentration
200-
- 30 .2_ 2
.20
1000
-40T~
exudaHon rote
3'0
6'0 9'0 120 1S0 exudation firne (rnin)
g
10~180 0
Fig. 2. Time course of sucrose concentration in the sieve-tube sap of Ricinus and exudation rate from the hypocotyl of cotyledons embedded in the endosperm. Seedlings were grown aseptically in water culture; the exudate was collected in 2-~tl samples
of sap was observed at the cut end of the hypocotyl. The exudation continued for 1-3 h with rates of between 15 pl and 40 p l - h - 1. The sucrose concentration in the exudate was 250 to 300 m M (Fig. 2) and stayed nearly constant for the whole exudation period. The concentration of exuded sucrose was more or less independent of the volume flow of exudation (Fig. 3) ; therefore, when exudation slowed down it did not affect the cOncentration of solutes in the exudate. Several criteria provide evidence that the exudate origina.'ted from sieve tubes: i) The exudate contained relatively
J. Kallarackal et al. : Sucrose transport into the sieve-tube of Ricinus
330
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E 400 wifh endosperm
300-
2
200-
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._c
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16
'
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310
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exudofion ro,fe {#['h -1" seedling -~)
,,,o
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exudofion fime(rain)
Fig. 3. Sucrose concentration in the sieve-tube sap of Ricinus seedlings exuding at different rates. The cotyledons were either embedded in the endosperm (filled symbols), or the cotyledons had been excised and incubated in buffer for 2 h before the hypocotyl was cut (open symbols)
Fig. 4. Sucrose concentration in the sieve-tube sap of exuding hypocotyls of Ricinus seedlings. The seedlings were grown in vermiculite, the endosperm was removed, the hypocotyl was cut and the cotyledons were immersed in 5 mM potassiumphosphate buffer pH 6.3. Different symbols indicate individual seedlings
high concentrations of sucrose (250-300mM), amino acids (130 raM) and K + (30 mM), but negligible concentrations of hexoses, ii) The pH of the exudate was 7.55. iii) The exudation could be stopped immediately by application of a small droplet of 0.1 M calcium chloride onto the hypocotyl stump, iv) No evidence for contamination of the exudate by xylem sap was obtained, because the incubation of cotyledons with the apoplastic marker PTS (Dybing and Currier 1961) did not lead to PTS in the exudate, v) Contamination of the exudate by the cell contents of the wounded parenchyma was negligible (with exception of the very first 2-pl sample), because the solute composition and the protein pattern of the exudate were constant over the whole exudation period and quantitatively and qualitatively different from those of the parenchyma extract of the hypocotyl.
externally supplied sugar. In some cases, however, the seedlings did not start to exude for I h or so after excision. It was found that the handling of the cotyledons during the removal of the endosperm could cause a reversible inhibition of phloem transport. Therefore the following procedure was routinely applied to seedlings: the endosperm was carefully removed from the cotyledons, which were then incubated in the medium for 2 h (the roots still immersed in 0.5 m M CaC12) before the hypocotyl was cut and exudation followed. Any further handling of the cotyledons, especially bending or squeezing was avoided.
Exudation from cotyledons without endosperm. The cotyledons of Ricinus seedlings take up solutes, which are supplied by the endosperm via the free space between the cotyledons and endosperm. Therefore it was of great interest to analyse the composition of sieve-tube sap from cotyledons, which had been excised from the endosperm and then incubated in medium without sugar. After removal of the endosperm, there was a steady decrease in the sucrose concentration in the sievetube sap until a steady level of 80-100 m M was reached after 2 h (Fig. 4). There was a slight tendency to find a higher sucrose concentration in those seedlings which exuded at a low rate (Fig. 3). These results show that the endogenous carbohydrate store of the cotyledons is either too small or not degraded fast enough to ensure continuously high sucrose exudation; therefore, the system would be expected to show a good response to
Sucrose in the sieve-tube sap during incubation of the cotyledons in media of different sucrose concentrations. When the cotyledons of seedlings from which the endosperm had been excised were incubated for 2 h in 5 m M potassium phosphate without sucrose or with different concentrations of sucrose, the exuded sieve-tube sap contained a constant concentration of sucrose throughout the exudation period (Fig. 5). In the absence of sucrose in the medium, the sucrose concentration in the sieve-tube sap was between 80 m M and I00 m M ; with sucrose in the incubation medium, the sucrose concentration of sieve-tube sap increased in a nonlinear manner (Fig. 6). At low levels in the medium there was a relatively steep increase of sucrose in the exudate; higher concentrations in the medium (i.e. above 50 raM) led to a further increase of sucrose in the sieve-tube sap, but the increase in concentration was at best the same as the increase in the medium. Half-maximal sucrose concentration in the sieve-tube sap was obtained at 2030 m M sucrose in the medium at which level sucrose was concentrated in the sieve-tube sap by
J. Kallarackal et al. : Sucrose transport into the sieve-tube of Ricinus
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exudation time (rain)
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Fig. 5. Time-course of sucrose concentration in the sieve-tube sap of exuding Ricinus seedlings, the cotyledons of which had
Fig. 7. Appearance of radioactive sucrose in the exudate from cut hypocotyls of Ricinus seedlings. Cotyledons of seedlings,
been preincubated for 2 h in media containing different sucrose concentrations. The cotyledons of seedlings, which had been grown in vermiculite and from which the endosperm had been excised, were incubated for 2 h in media containing the indicated sucrose concentrations. The hypocotyls then were cut and exudate samples were collected while the cotyledons were still incubated in the sucrose media
which had been grown in water culture and from which the endosperm had been removed, were preincubated fi)r 2 h in 50 m M sucrose, which was either labelled with [~4C]sucrose (closed symbols) or unlabelled (open symbols). In this latter case, [14C]sucrose was added when the hypocotyls were cut at the end of the 2-h preincubation period
~S
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Fig. 6. Dependence of the sucrose concentration in the sievetube sap of Ricinus seedlings on the sucrose concentration in the medium. Cotyledons of seedlings, which had been grown in water culture, were incubated for 2 h in media containing different sucrose concentrations. The hypocotyls were then cut and the exudate collected
u ~
/// /
2000 "s
-b--
3-
/
1
/ o//' / /
2 a factor of 7-10; however, accumulation was even observed at the highest ambient sucrose levels. Time-course o f sucrose flow from the medium to the sieve tube. When the medium bathing the cotyledons was labelled with [l~C]sucrose during exudation, radioactivity appeared in the sieve-tube sap (Fig. 7) after a time lag of approx. 10 min. After about 2 h, the sieve-tube sap had the same concentration of radioactivity that was found in plants, which had received [~4C]sucrose during the whole preincubation period. The time-course of exudation of radioactivity was sensitive to osmotic changes in a complex manner. When the radioac-
~
o/
1-
0
~" B ~"'S /
S ~'S
/
J
r -4r
J0
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exudation time (rain)
Fig. 8. Time-course of volume flow and of [14C]sucrose exudation from the hypocotyls of Ricinus seedlings. Before the addition of labelled sucrose the cotyledons had been incubated in buffer or sucrose. The cotyledons of seedlings, which had been grown in water culture and from which the endosperm had been removed, were incubated for 2 h either in 5 m M buffer without sucrose ~ - -) or with i00 m M sucrose (---). 1"hen the hypocoty;s were cut and exudation was allowed for" 60 rain. Then all the cotyledons were transferred to 100 m M [~4C]sucrose. S ~ * S indicates seedlings which were transferred from 1 0 0 r a M sucrose to 1 0 0 r a M [t4C]sucrose; B ~ + S indicates seedlings which were transferred from buffer to 100raM [l~C]sucrose
332 tive sucrose was a d d e d to seedlings which had alr e a d y been i n c u b a t e d in sucrose for 2 h, then a steadily increasing a m o u n t o f label a p p e a r e d in the sieve-tube sap (Fig. 8). W h e n the seedlings h a d been p r e i n c u b a t e d in buffer before the m e d i u m was labelled with sucrose, then the v o l u m e flow stopped for some time and then resumed after 60 min. Despite this transient cessation o f exudation, the sieve-tube sap, which was eventually exuded actually c o n t a i n e d a slightly higher concentration o f radioactivity t h a n the samples f r o m the c o n t i n u o u s l y exuding seedlings. The total a m o u n t o f radioactivity, which was exuded was, within the first hour, similar in b o t h sets o f seedlings despite the small v o l u m e flow in those which were transferred f r o m buffer to sucrose. In these latter seedlings, it is obvious t h a t during the period w i t h o u t e x u d a t i o n the sieve tubes in the cotyledons were loaded just as well as in those seedlings, which had r e m a i n e d at a c o n s t a n t sucrose concentration, even t h o u g h the water gain into the sieve tubes and therefore the e x u d a t i o n o f sieve-tube sap was delayed for some time by the increase in osmotic strength o f the medium. This result shows that sugar translocation within the sieve tubes r e s p o n d e d t o osmotic changes, whereas sugar loading into the sieve tubes did not. (The larger a m o u n t o f exudation o f label in the buffer-pretreated seedling after 2 h could have been the result o f a larger specific radioactivity o f sucrose in these seedlings, because the level o f non-labelled sucrose in the cotyledons had been lower at the time o f transfer to labelled sucrose.)
Relationship between the K + concentration in the sieve-tube sap and sugar transport. W h e n cotyledons f r o m which the e n d o s p e r m h a d been r e m o v e d were i n c u b a t e d in 5 m M p o t a s s i u m - p h o s p h a t e buffer, a steady e x u d a t i o n o f K +-containing sievetube sap was observed. The K + c o n c e n t r a t i o n seemed m o s t l y i n d e p e n d e n t o f the e x u d a t i o n rate in the range o f 5-20 p l . h 1; a slight increase o f K + was observed at very low e x u d a t i o n rates (data n o t shown). W h e n the c o n c e n t r a t i o n o f sucrose was varied between 100 and 400 m M in the sievetube sap by i n c u b a t i o n o f the c o t y l e d o n s in media o f various c o n c e n t r a t i o n s o f sucrose, no obvious change in the K + c o n c e n t r a t i o n in the sieve-tube sap o c c u r r e d (Fig. 9). At c o n s t a n t sucrose c o n c e n t r a t i o n in the mediu m and varying c o n c e n t r a t i o n s o f K + the m o s t obvious effect o f K + was on the e x u d a t i o n rate. T h e r e was consistently an extremely low e x u d a t i o n rate at high K + c o n c e n t r a t i o n s (Fig. 10). T h e sucrose c o n c e n t r a t i o n in the sieve-tube sap was n o t
J. Kallarackal et al. : Sucrose transport into the sieve-tube of Ricinus
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9
~80
9
~
9e
9
9
9
9
.~ 60~>
40-
~c 20~" o
o
16o 260 360 460 s6o 660 = sucrose in sieve tube sap (raM)
Fig. 9. Concentration of K + in the sieve-tuve sap of Ricinus seedlings at different concentrations of sucrose in the sieve-tuve sap. The cotyledons of seedlings, grown in vermiculite, were incubated in 5 mM potassium phosphate and various sucrose concentrations (0-200 mM) for 2 h. Then, exudation was started by cutting the hypocotyls and the exudate was analyzed for sucrose and K +
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,~ 20010 2
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o
io
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Fig. 10. Dependence of the exudation rate and the sucrose concentration in the sieve-tube sap of Ricinus seedlings on the K + concentration in the medium. The cotyledons of water-cultivated seedlings, from which the endosperm had been removed, were incubated in 50 mM sucrose for 2 h in media containing the indicated concentrations of K + (as phosphate). Then the hypocotyls were cut and exudation was followed
affected by K +, t h o u g h there a p p e a r e d to be a slight t e n d e n c y towards b o t h a higher flow rate and a higher sucrose c o n c e n t r a t i o n at approx. 1020 m M K +, which in c o m b i n a t i o n resulted in a 70% higher rate o f sucrose t r a n s l o c a t i o n at that K + c o n c e n t r a t i o n in the medium.
Estimation of the in-vivo rate of phloem transport and of the translocation speed. Cutting the h y p o c o t yl o f the Ricinus seedlings in order to obtain p h l o e m exudate r e m o v e d the sink tissues, namely the lower part o f the h y p o c o t y l and the r o o t system, and it is n o t k n o w n whether these tissues controlled p h l o e m t r a n s p o r t by limiting assimilate c o n s u m p t i o n . On the o t h e r hand, the slicing process could cause plugging o f some sieve tubes, thereby decreasing the rate o f solute flow far below that in the intact seedling. Therefore, a realistic estimate o f the in-vivo rate o f p h l o e m t r a n s p o r t
J. Kallarackal et al. : Sucrose transport into the sieve-tube of Ricinus Table 1. Dry weight of organs of Ricinus seedlings from days
4 to 6. The seedlings were grown in vermiculite for 4~6 d and ten seedlings were harvested per day, weighed, dried for 24 h at 80 ~ and weighed again. The SD was between 7 and 12% of the mean value. Respiration was measured with an oxygen electrode. The average seedling as used for the experiments was at the age of 5-6 d Dry weight (mg. (seedling)- 1)
Endosperm Cotyledons Hypocotyl Roots
day4
day5
283 32 22 21
246 43 33 34
Dry-weight change from day5 6 d a y 6 (mg.h ~ 9(seedling) - 1)
Respiration (mg sucrose .h 1(seed_ ling)-1)
88 90 109 46
-0.25 0.03} 0.23 0.26
--6.58 1.96 3.16} 0.5 3.66
had to be made to compare it with the rates of the exuding system. The net rate of phloem transport was calculated from the increase in dry weight of the hypocotyl and roots plus the respiratory loss of carbon by these organs (Table 1). The total dry-matter transfer through the phloem from the cotyledons to the hypocotyl amounted to 3.92 ragh - 1. (seedling)- 1. The rates of exudation of sievetube sap of seedlings, whose cotyledons were embedded in the endosperm varied between 20 and 40 gl-h 1, with the average at 30 pl.h -1. Since the sieve-tube sap contained 300 m M sucrose, 130 m M amino acids, 30 m M K + and probably 30 m M organic anions, a flow rate of 31 pl.h -1 would be required to account for the in-vivo net mass-transfer rate. We therefore conclude that the experimental system is not artificial in the sense that it causes entirely unnatural flow rates (Kallarackal and Milburn 1984). From the flow rate in vivo and during exudation the speed of phloem transport in the hypocotyl could be calculated. The Ricinus seedling has eight conducting bundles in the hypocotyl at the place of cutting, and these eight bundles together contain sieve tubes with a cross-sectional area of 0.030_+ 0.011 mm 2. A volume flow of 31 gl.h -1 means, if all sieve tubes are operating equally well, a flow speed of 1.03 m . h -1 or 12 r a m . r a i n - I ; the fastest exudation rate measured in a seedling was 47 pl. h-1, which corresponds to a flow speed of 1.6 m. h -1"
Discussion
There are several arguments, both theoretical and experimental, that the sap bleeding from the cut hypocotyl really stemmed from the sieve tubes. The
333
exudate consistently contained sucrose concentrations five- to ten-times higher than the bathing medium, and other solutes e.g. potassium and amino acids were also found to be concentrated. The p H of the sap was 7.55, a value close to the usually observed p H of sieve-tube sap. 3-Hydroxy-5,8,10pyrenetrisufonate, which is generally regarded as an apoplast marker (Dybing and Currier 1961), did not show up in the exuding sap when added to the medium, so that even minor contamination (2%) of the sieve-tube exudate with apoplast solutes can be ruled out within measuring accuracy. The exudation rate of cut seedlings, which still contained endosperm, was usually the same as that calculated from the growth increment for the net flow in the intact seedling. The mean speed of solvent flow, 1.03 m . h -1, is normal in the light of more recent literature (Minchin and Troughton 1980). One serious problem is the fact that the percentage of open sieve tubes after cutting is not controlled. It could be envisaged that where only a few sieve tubes are open the solutes mJ[ght be directed into these few conduits, resulting in a solute concentration larger than natural. :The comparison of volume flow rates and sucrose concentrations under otherwise identical experimental conditions showed that within the measured range there is no interdependence of these parameters. Results from seedlings with different flow rates are therefore comparable. It is unlikely that cutting the hypocotyl caused a surge or an increase in volume flow or an artificial change of solute concentration or solute composition. The first appearance of label in the exudate is noted within 10 rain after addition of label to the medium, but the uniform labelling of sieve-tube sap took up to 2 h; therefore, it appears that it is not the transfer of solutes from the medium to the sieve tubes but the uniform labelling of endogenous carbohydrate pools that is a slow process. The concentration of sucrose in the sieve-tube sap under natural conditions, with the endosperm attached is high, 250-300 raM, though not as high as measured with adult Ricinus (Smith and Milburn 1980 a-c), probably because water availability in the leaves of adult plants is always less than in the seedling. The concentration of sucrose in the sap and the exudation rate clearly behaved as in an osmotic system, as was obvious when an osmotic change in the medium occurred during exudation. Because the apoplastic concentration of solutes was probably identical to that of the medium it can safely be stated that at a particular apoplastic concentration the sieve-tube sap has a particular,
334 definite solute content. Thus, for the first time, a concentration dependence of sucrose loading into the phloem could be measured. At 100 m M sucrose in the medium the sucrose concentration in the sieve tubes has the same value as was determined using endosperm-attached cotyledons. The measured sucrose concentration at the endosperm-cotyledon interface was 70-90 m M (Kriedemann and Beevers 1967a; Komor 1977). An obvious difference between our seedling system and the few known systems with adult plants is the ease with which the sucrose concentration in the sieve-tube sap can be varied by altering the supply of external sucrose. For instance in adult plants the day-night rhythm showed little variation (Smith and Milburn 1980a), whereas in the seedlings the sucrose concentration in sievetube sap decreased to one-fourth within 2 h when internal, stored carbohydrates were the only source. For many years, the effect of K § on sucrose loading has been a common topic in the literature (Mengel and H/ider 1977; Lang 1979). Usually the correlation between sucrose and K + concentration was determined, since it was not really possible to apply defined concentrations of K § to the apoplast and to monitor simultaneously the sucrose transport into the sieve tubes. The Ricinus seedling system has allowed this experiment to be carried out for the first time, and has demonstrated that there is a pronounced effect of K § application on the K + content of sieve-tube sap (data not shown) but definitely not on the sucrose content. If there is a promotion of phloem loading by K § then either it occurs prior to an apoplastic loading step, for instance at the exit of sucrose from the mesophyll, or the very low concentration of K § found in situ is already sufficient for maximal activity. What are the advantages and disadvantages of the exuding Ricinus seedling system compared with other known systems for the analysis of sieve-tube sap (Ziegler 1975)? i) The Ricinus seedling usually exudes sieve-tube sap only for 2 3 h, but that period can be prolonged by further slicing. Much longer periods have been reported for incisions of adult plants or for the cut stylets of aphids, (though very short exudation periods can also occur with the latter), ii) The sample volumes are smaller than from cut adult Ricinus, but are by far larger than from aphid stylets. Compared with the latter, chemical analysis is much easier and evaporation is a much smaller problem, iii) The percentage of open sieve tubes after cutting is unknown, and in this respect the situation is similar to that of incised adult plants (and to aphid experi-
J. Kallarackalet al. : Sucrose transport into the sieve-tubeof Ricinus ments, too, where partial plugging is possible). Therefore it is not possible to draw conclusions about mass-transfer rates in a single, isolated experimental situation, iv) Cutting the hypocotyl of the seedling does not increase the flow rate of sievetube sap. Therefore, artificial effects caused by a change in the water flow do not have to be taken into account. In adult plants, incised or infested with aphids, this is a serious problem, v) Both the exuding Ricinus seedling and the incision of adult plants allow the determination of the real concentration of compounds in the sieve-tube sap. This is in contrast to the leakage caused by ethylenediaminetetraacetic acid (King and Zeevart 1974), and also to the aphid method, where considerable evaporation has to be envisaged because of the minute exudate volumes, vi) The exuding Ricinus seedling is the only system where the uptake o f ' particular compounds by the leaves, either natural compounds or xenobiotics, and the composition of sieve-tube sap can be monitored simultaneously. Since the compounds can be offered at defined concentrations to the apoplast, a quantitative analysis of the concentration dependence of phloem loading for that particular compound is possible. Future experiments with the Ricinus seedling may help to determine which factors regulate phloem loading, which solutes besides sucrose can be loaded into the sieve tubes, and which concentrations of particular solutes are achieved in the sieve tubes when the concentration and conditions in the apoplast are defined. During revision of this manuscript, a report appeared describing experiments involving exudation from the cut hypocotyl of Ricinus seedlings (Vreugdenhil and Koot-Gronsveld 1988). Only seedlings with the endosperm attached were used, and their results were essentiallythe same as those reported by us in the first section of the Results. The experimentalhelp given by Klaus Wassermannand Walter K6ckenberger, and the financial funding by Deutsche Forschungsgemeinschaftand by A.v. Humboldt-Stiftung(to Jose Kallarackal) are gratefullyacknowledged References Daie, J. (1987) Sucrose uptake in isolated phloem segments of celery is a single saturable transport system. Planta 171, 474482 Dybing, D.C., Currier, H.B. (1961) Foliar penetration by chemicals. Plant Physiol.36, 169 174 Fisher, D.B. (1981) Measurement of the sieve tube membrane potential. Plant Physiol.67, 845 849 Hall, S.M., Baker, D.A., Milburn, J.A. (1971) Phloemtransport of 14C-labelledassimilatesin Rieinus. Planta 100, 200-207 Kallarackal, J., Milburn, J.A. (1984) Specificmass transfer and sink-controlled phloem translocation in castor bean. Aust. J. Plant Physiol. 11,483~490
J. Kallarackal et al. : Sucrose transport into the sieve-tube of Ricinus King, R.W., Zeevart, J.A.D. (1974) Enhancement of phloem exudation from cut petioles by chelating agents. PIant Physiol. 53, 96-103 Komor, E. (1982) Transport of sugar. In: Encyclopedia of ptant physiology, N.S., vol. :I3A Plant Carbohydrates I. IntracelIular Carbohydrates, pp. 635-676, Loewus, F.A., Tanner, W., eds., Springer, Berlin Heidelberg Komor, E. (1977) Sucrose uptake by cotyledons of Ricinus communis L. Characteristics, mechanism and regulation. Planta 137, 119-131 Kriedemann, P., Beevers, H. (1967 a) Sugar uptake and transtocation in the castor bean seedling. I. Characteristics of transfer in intact and excised seedlings. Plant Physiol. 42, 161-173 Kriedemann, P., Beevers, H. (1967 b) Sugar uptake and translocation in the castor bean seedling. II. Sugar transformations during uptake. Plant Physiol. 42, 17z1~180 Kursanov, A.L. (1984) Assimilate transport in plants. Elsevier, Amsterdam Lang, A. (1979) A relay mechanism for phloem translocation. Ann. Bot. 44, 141-I45 Mengel, K., H/ider, H.E. (1977) Effect of potassium suppIy on the rate of phloem sap exudation and the composition of phloem sap of Ricinus communis. Plant Physiol. 59, 282284 Minchin, P.E., Troughton, J.H. (1980) Quantitative interpretation of phloem translocation data. Annu. Rev. Plant Physiol. 31, 191-215
335 Schobert, C., Komor, E. (1987) Amino acid uptake by Ricinus communis roots: characterization and physiologicaI significance. Plant Cell Environ. 10, 493 500 Smitb, J.A.C., Milburn, J.A. (1980a) Osmoregulation and the control of phloem-sap composition in Ricinus communis L. Planta 148, 28 34 Smith, J.A.C., Milburn, J.A. (1980b) Phloem transport, solute flux and the kinetics of sap exudation in Ricinus communis L. Planta 148, 35-41 Smith, J.A.C., Milburn, J.A. (1980c) Phloem turgot and the regulation of sucrose loading in Ricinus communis L. Planta 148, 42~48 Sovonick, S.A., Geiger, D.R., Fellows, R.J. (1974) Evidence for active phloem loading in the minor veins Of sugar beet. Plant Physiol. 54, 886-891 Vreugdenhil, D., Koot-Gronsveld, E.A.M. (1988) Characterization of phloem exudation from castor-bean cotyledons. Planta 174, 38(~384 Yemm, E.W., Willis, A.J. (1954) The estimation of carbohydrates in plant extracts by anthrone. Biochem. J. 57, 508 514 Ziegler, H. (1975) Nature of transported substances. In: Encyclopedia of plant physiol., N.S., vol. 1 : Transpbrt in plants I. Phloem transport pp. 59-100 Zimmerman~ M.H. Milburn, J.D. eds., Springer, Berhn Heidelberg New York Received 9 March: accepted 5 October 1988
P l a n t a 9 Springer-Verlag 1989
Sucrose transport into the phloem of Ricinus communis L. seedlings as measured by the analysis of sieve-tube sap Jose Kallarackal*, Gabriele Orlich, Christian Schobcrt, and Ewald Komor** Pflanzenphysiologie, Universit/it Bayreuth, Postfach 1012 51, D-8580 Bayreuth, Federal Republic of Germany
Abstract. Careful cutting of the hypocotyl of R i c i nus c o m m u n i s L. seedlings led to the exudation of pure sieve-tube sap for 2-3 h. This offered the possibility of testing the phloem-loading system qualitatively and quantitatively by incubating the cotyledons with different solutes of various concentrations to determine whether or not these solutes were loaded into the sieve tubes. The concentration which was achieved by loading and the time course could also be documented. This study concentrated on the loading of sucrose because it is the major naturally translocated sieve-tube compound. The sucrose concentration of sieve-tube sap was approx. 300 m M when the cotyledons were buried in the endosperm. When the cotyledons were excised from the endosperm and incubated in buffer, the sucrose concentration decreased gradually to 80-100 mM. This sucrose level was maintained for several hours by starch breakdown. Incubation of the excised cotyledons in sucrose caused the sucrose concentration in the sieve tubes to rise from 80 to 400 mM, depending on the sucrose concentration in the medium. Thus the sucrose concentration in the sieve tubes could be manipulated over a wide range. The transfer of labelled sucrose to the sieve-tube sap took I0 min; full isotope equilibration was finally reached after 2 h. An increase of K § in the medium or in the sieve tubes did not change the sucrose concentration in the sievetube sap. Similarly the experimentally induced change of sucrose concentration in the sieve tubes did not affect the K § concentration in the exudate. High concentrations of K § however, strongly re* P r e s e n t a d d r e s s : Plant Physiology Division, Kerala Forest Research Institute, Peechi 680 653, Trichur, Kerala, India ** To whom correspondence should be addressed A b b r e v i a t i o n ." PTS
= 3-hydroxy-5,8,J 0-pyrenetrisulfonate
duced the flow rate of exudation. Similar results were obtained with Na + (data not shown). The minimum translocation speed in the sieve tubes in vivo was calculated from the growth increment of the seedling to be 1.03 m - h 1, a value, which on average was also obtained for the exudation system with the endosperm attached. This comparison of the in-vivo rate of phloem transport and the exudation rate from cut hypocotyls indicates that sink control of phloem transport in the seedlings of that particular age was small, if there was any at all, and that the results from the experimental exudation system were probably not falsified by removal of the sink tissues.
Key words: Exudation (phloem, K + effect) Phloem (translocation speed) Potassium (phloem) - R i c i n u s (phloem transport) - Sink control - Sucrose loading
Introduction The transport of solutes by the phloem is an :important step in the partitioning of assimilates in the plant. However, the mechanism and the specificity of the transport of solutes into the sieve tubes are not known because the complexity of the tissue has not allowed unambiguous kinetic measurements, as is possible for example in homogeneous parenchyma tissues and single cells (reviewed by K o m o r 1982; Kursanov 1984). So far the investigations of phloem loading have been of two types. One approach was the analysis of sieve-tube sap from those (few) plants which either exhibit "phloem bleeding" upon incision of the bark (Hall et al. ]971 ; Smith and Milburn 1980a-c) or which
328
could be infested with aphids, whose stylets could then be cut (Fisher 1981). In this way the composition of sieve-tube sap in plants under natural conditions has been determined. These systems were, however, limited by the difficulty of manipulating, both qualitatively and quantitatively, the composition of the exudate: incubation of the leaf veins was difficult because of the cuticle, and the storage compounds in the leaf "buffered" most of the experimental treatments. For example, darkening the leaves for some hours hardly affected the sucrose concentration in the exuding sap of Ricinus (Smith and Milburn 1980 a). Furthermore, because solutes cannot be applied in definite concentrations to the leaf veins, it could not be decided whether the lack of a particular substance in the sieve-tube sap was because of the unavailability of this substance at the phloem-loading site or because of the incapability of the loading system to take up this substance. The other common type of experiment involved the use of pieces of phloem strands or leaf discs (Sovonick et al. 1974; Daie 1987) which were usually incubated with labelled substances and the uptake of label determined and localized by autoradiography. These studies suffered from the disadvantage that it was difficult to prove that the label, which for instance was found in the veins, was still in the compound which had been supplied and was not in a metabolic product. In addition the labelling of the veins cannot necessarily be considered to be identical to the labelling of the sieve tubes. Therefore, we have tried to elaborate an experimental system, which combines the advantages of the analysis of sieve-tube sap with those of the incubation method, with the aim of investigating the response of the sieve-tube loading system to defined conditions in the leaf. We chose the Ricinus seedling as a model system because the cotyledons, since they do not have a cuticle, readily respond to incubation with solutes (Kriedemann and Beevers 1967a, b). Since the adult Ricinus plant is one of the few, plants, which show phloembleeding after incision, we hoped that cutting or incising the hypocotyl of the seedling might give rise to sieve-tube exudation. With this system it would be possible to follow the loading of substances of interest into the sieve tubes and to reveal which conditions and factors regulate solute transport into the phloem. This report demonstrates that pure sieve-tube sap can be obtained from Ricinus seedlings for several hours after cutting the hypocotyl and that the seedling responds to experimental changes both rapidly and extensively.
J. Kallarackal et al. : Sucrose transport into the sieve-tube of Ricinus
Material and methods Plant material and chemicals'. Seeds of Ricinus communis L. cv. gibsonii were either obtained from greenhouse-grown plants or were purchased from Walz, Stuttgart, F R G . The 14C-labelled compounds were bought from Amersham, Braunschweig (FRG), and N E N - D u p o n t , Dreieich (FRG). 3-Hydroxy-5,8,10pyrenetrisulfonate (PTS) was a gift from Dr. J.J. Jachetta (Cornell University, Ithaca, N.Y.). Growth of seedlings. The seeds were immersed in 0.3% chinosol solution for i0 min and then soaked in water overnight. Initially, they were planted in wet vermiculite the following day, incubated in a humid atmosphere at 27~ in dim light for 5 d, and seedlings of average size then were selected for the experiments. Later, it was found that a better yield of exuding seedlings could be obtained with plants, which were raised axenically in water culture. In that case the seed coat was removed and the naked seed was surface-sterilized in hydrogen peroxide (Schobert and K o m o r 1987). After repeated washings the seeds were germinated for 2 d on agar enriched in organic nutrients to ensure the absence of bacteria. Then the seedlings were transferred to a sterile aerated water culture and were allowed to grow up to the sixth day.
Incubation, cutting and collection of sieve-tube sap. When the seedlings were grown in vermiculite, they were not removed from the substratum, but kept there until the hypocotyls were cut. Water-cultivated seedlings were carefully transferred to a beaker with the roots immersed in 0.5 m M CaC12 at 27 ~ C. The individual steps of the preparation, cutting and sap collection are illustrated in Fig. 1. Depending on the intention of the investigation, the endosperm was carefully removed without bending or squeezing the cotyledons or the hypoeotyl, Then the cotyledons were incubated in buffer solution, e.g. 5 m M potassium phosphate pH 6.3, or in buffer supplemented with sugar, ions or other compounds of interest (the roots still immersed in 0.5 m M CaC12). For exudation the hypocotyl was severed in the hook region approx. 10 m m apart from the cotyledons with a sharp razor blade, using a steady, non-squeezing motion. The time-point of cutting was either at the moment of addition of a particular solute, or after 2 h of incubation with the solute. After cutting, the exudation of sap from the cut end of the upright hypocotyl was observed; in those cases where no exudation occurred, a further slicing of the hypocotyl helped to initiate exudation. The cut hypoeotyl was leaning against the wall of a small beaker or was supported with putty. The sap was usually collected continuously with graded glass capillaries until a 2-gl sample was obtained; the time-point of collection was noted and the 2-I,tl sample was immediately diluted with water to 200 lal and was kept in the cold till analysis. In order to prevent evaporation from the droplet on the exuding hypocotyl stump, the whole incubation was performed in a water bath at 27 ~ C under a Plexiglas hood, which was layed out with wet filter paper. Small openings at the side of the hood allowed insertion of the microcapillaries for exudate collections. Evaporation was experimentally determined by putting a droplet with a defined sucrose concentration onto the stump of a non-exuding seedling. We found that evaporation caused the sucrose concentration to increase by less than 3% within 5 min, and a droplet never remained longer than 5 min on the cut stump. Evaporation from the microcapillaries was negligible (below 2% in 10 rain).
Analytical methods. Sugars, i.e. sucrose, glucose and fructose, were quantified enzymatically with a test set from Boehringer, Mannheim, F R G , or by the anthrone method (Yemm and Wit-
J. Kallarackal et aI. : Sucrose transport into the sieve-tube of Ricinus
Fig. 1 A-E. Illustration of the seedling preparation, cutting and
sieve tube sap collection. A Six days old Ricinus seedlings before (left) and after (right) endosperm removal. B Preincubation of the cotyledons in a defined solution with the roots immersed in 0.5 m M CaC12. The arrow indicates the site of cutting. C Incubation of the cotyledons during exudation. D Sieve tube sap exudation from the hypocotyl stump. E Exudate collection with microcapiilary under the hood of the temperature-controlled water bath
lis 1954). SampIes were chromatographed on thin-layer cellulose gels with ethyl acetate-pyridine-water, 100:35:25 (by vol.) as solvent. The inorganic ions such as K § Na § Ca 2+ and Mg 2+ were determined by atomic absorption (Perkin-Elmer 5000, Norwalk, Conn., USA). The presence of the apoplastic marker 3-hydroxy-5,8,10-pyrenetrisulfonate (PTS) (Dybing and Currier 1961) in the phloem exudate (20-gl samples) was measured in microcuvettes using a fluorescence spectrometer (Perkin-Elmer 650 10 M) at 405-nm excitation and 510-nm emission wavelengths. Radioactivity was measured by scintillation counting in dioxan-naphthalene-diphenyloxazol (1000:100: 5 by vol.). The pH of sieve-tube sap was determined with a flat-bottomed electrode (Ingold, Ziirich, Switzerland) in 10-gl samples.
Results
Establishment o f the exudation system with the endosperm attached to the cotyledons. When the hypocotyl of a 6-d-old Ricinus seedling was cut near the cotyledons just below the hook, an exudation
329
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Fig. 2. Time course of sucrose concentration in the sieve-tube sap of Ricinus and exudation rate from the hypocotyl of cotyledons embedded in the endosperm. Seedlings were grown aseptically in water culture; the exudate was collected in 2-~tl samples
of sap was observed at the cut end of the hypocotyl. The exudation continued for 1-3 h with rates of between 15 pl and 40 p l - h - 1. The sucrose concentration in the exudate was 250 to 300 m M (Fig. 2) and stayed nearly constant for the whole exudation period. The concentration of exuded sucrose was more or less independent of the volume flow of exudation (Fig. 3) ; therefore, when exudation slowed down it did not affect the cOncentration of solutes in the exudate. Several criteria provide evidence that the exudate origina.'ted from sieve tubes: i) The exudate contained relatively
J. Kallarackal et al. : Sucrose transport into the sieve-tube of Ricinus
330
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Fig. 3. Sucrose concentration in the sieve-tube sap of Ricinus seedlings exuding at different rates. The cotyledons were either embedded in the endosperm (filled symbols), or the cotyledons had been excised and incubated in buffer for 2 h before the hypocotyl was cut (open symbols)
Fig. 4. Sucrose concentration in the sieve-tube sap of exuding hypocotyls of Ricinus seedlings. The seedlings were grown in vermiculite, the endosperm was removed, the hypocotyl was cut and the cotyledons were immersed in 5 mM potassiumphosphate buffer pH 6.3. Different symbols indicate individual seedlings
high concentrations of sucrose (250-300mM), amino acids (130 raM) and K + (30 mM), but negligible concentrations of hexoses, ii) The pH of the exudate was 7.55. iii) The exudation could be stopped immediately by application of a small droplet of 0.1 M calcium chloride onto the hypocotyl stump, iv) No evidence for contamination of the exudate by xylem sap was obtained, because the incubation of cotyledons with the apoplastic marker PTS (Dybing and Currier 1961) did not lead to PTS in the exudate, v) Contamination of the exudate by the cell contents of the wounded parenchyma was negligible (with exception of the very first 2-pl sample), because the solute composition and the protein pattern of the exudate were constant over the whole exudation period and quantitatively and qualitatively different from those of the parenchyma extract of the hypocotyl.
externally supplied sugar. In some cases, however, the seedlings did not start to exude for I h or so after excision. It was found that the handling of the cotyledons during the removal of the endosperm could cause a reversible inhibition of phloem transport. Therefore the following procedure was routinely applied to seedlings: the endosperm was carefully removed from the cotyledons, which were then incubated in the medium for 2 h (the roots still immersed in 0.5 m M CaC12) before the hypocotyl was cut and exudation followed. Any further handling of the cotyledons, especially bending or squeezing was avoided.
Exudation from cotyledons without endosperm. The cotyledons of Ricinus seedlings take up solutes, which are supplied by the endosperm via the free space between the cotyledons and endosperm. Therefore it was of great interest to analyse the composition of sieve-tube sap from cotyledons, which had been excised from the endosperm and then incubated in medium without sugar. After removal of the endosperm, there was a steady decrease in the sucrose concentration in the sievetube sap until a steady level of 80-100 m M was reached after 2 h (Fig. 4). There was a slight tendency to find a higher sucrose concentration in those seedlings which exuded at a low rate (Fig. 3). These results show that the endogenous carbohydrate store of the cotyledons is either too small or not degraded fast enough to ensure continuously high sucrose exudation; therefore, the system would be expected to show a good response to
Sucrose in the sieve-tube sap during incubation of the cotyledons in media of different sucrose concentrations. When the cotyledons of seedlings from which the endosperm had been excised were incubated for 2 h in 5 m M potassium phosphate without sucrose or with different concentrations of sucrose, the exuded sieve-tube sap contained a constant concentration of sucrose throughout the exudation period (Fig. 5). In the absence of sucrose in the medium, the sucrose concentration in the sieve-tube sap was between 80 m M and I00 m M ; with sucrose in the incubation medium, the sucrose concentration of sieve-tube sap increased in a nonlinear manner (Fig. 6). At low levels in the medium there was a relatively steep increase of sucrose in the exudate; higher concentrations in the medium (i.e. above 50 raM) led to a further increase of sucrose in the sieve-tube sap, but the increase in concentration was at best the same as the increase in the medium. Half-maximal sucrose concentration in the sieve-tube sap was obtained at 2030 m M sucrose in the medium at which level sucrose was concentrated in the sieve-tube sap by
J. Kallarackal et al. : Sucrose transport into the sieve-tube of Ricinus
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Fig. 7. Appearance of radioactive sucrose in the exudate from cut hypocotyls of Ricinus seedlings. Cotyledons of seedlings,
been preincubated for 2 h in media containing different sucrose concentrations. The cotyledons of seedlings, which had been grown in vermiculite and from which the endosperm had been excised, were incubated for 2 h in media containing the indicated sucrose concentrations. The hypocotyls then were cut and exudate samples were collected while the cotyledons were still incubated in the sucrose media
which had been grown in water culture and from which the endosperm had been removed, were preincubated fi)r 2 h in 50 m M sucrose, which was either labelled with [~4C]sucrose (closed symbols) or unlabelled (open symbols). In this latter case, [14C]sucrose was added when the hypocotyls were cut at the end of the 2-h preincubation period
~S
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Fig. 6. Dependence of the sucrose concentration in the sievetube sap of Ricinus seedlings on the sucrose concentration in the medium. Cotyledons of seedlings, which had been grown in water culture, were incubated for 2 h in media containing different sucrose concentrations. The hypocotyls were then cut and the exudate collected
u ~
/// /
2000 "s
-b--
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/
1
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2 a factor of 7-10; however, accumulation was even observed at the highest ambient sucrose levels. Time-course o f sucrose flow from the medium to the sieve tube. When the medium bathing the cotyledons was labelled with [l~C]sucrose during exudation, radioactivity appeared in the sieve-tube sap (Fig. 7) after a time lag of approx. 10 min. After about 2 h, the sieve-tube sap had the same concentration of radioactivity that was found in plants, which had received [~4C]sucrose during the whole preincubation period. The time-course of exudation of radioactivity was sensitive to osmotic changes in a complex manner. When the radioac-
~
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exudation time (rain)
Fig. 8. Time-course of volume flow and of [14C]sucrose exudation from the hypocotyls of Ricinus seedlings. Before the addition of labelled sucrose the cotyledons had been incubated in buffer or sucrose. The cotyledons of seedlings, which had been grown in water culture and from which the endosperm had been removed, were incubated for 2 h either in 5 m M buffer without sucrose ~ - -) or with i00 m M sucrose (---). 1"hen the hypocoty;s were cut and exudation was allowed for" 60 rain. Then all the cotyledons were transferred to 100 m M [~4C]sucrose. S ~ * S indicates seedlings which were transferred from 1 0 0 r a M sucrose to 1 0 0 r a M [t4C]sucrose; B ~ + S indicates seedlings which were transferred from buffer to 100raM [l~C]sucrose
332 tive sucrose was a d d e d to seedlings which had alr e a d y been i n c u b a t e d in sucrose for 2 h, then a steadily increasing a m o u n t o f label a p p e a r e d in the sieve-tube sap (Fig. 8). W h e n the seedlings h a d been p r e i n c u b a t e d in buffer before the m e d i u m was labelled with sucrose, then the v o l u m e flow stopped for some time and then resumed after 60 min. Despite this transient cessation o f exudation, the sieve-tube sap, which was eventually exuded actually c o n t a i n e d a slightly higher concentration o f radioactivity t h a n the samples f r o m the c o n t i n u o u s l y exuding seedlings. The total a m o u n t o f radioactivity, which was exuded was, within the first hour, similar in b o t h sets o f seedlings despite the small v o l u m e flow in those which were transferred f r o m buffer to sucrose. In these latter seedlings, it is obvious t h a t during the period w i t h o u t e x u d a t i o n the sieve tubes in the cotyledons were loaded just as well as in those seedlings, which had r e m a i n e d at a c o n s t a n t sucrose concentration, even t h o u g h the water gain into the sieve tubes and therefore the e x u d a t i o n o f sieve-tube sap was delayed for some time by the increase in osmotic strength o f the medium. This result shows that sugar translocation within the sieve tubes r e s p o n d e d t o osmotic changes, whereas sugar loading into the sieve tubes did not. (The larger a m o u n t o f exudation o f label in the buffer-pretreated seedling after 2 h could have been the result o f a larger specific radioactivity o f sucrose in these seedlings, because the level o f non-labelled sucrose in the cotyledons had been lower at the time o f transfer to labelled sucrose.)
Relationship between the K + concentration in the sieve-tube sap and sugar transport. W h e n cotyledons f r o m which the e n d o s p e r m h a d been r e m o v e d were i n c u b a t e d in 5 m M p o t a s s i u m - p h o s p h a t e buffer, a steady e x u d a t i o n o f K +-containing sievetube sap was observed. The K + c o n c e n t r a t i o n seemed m o s t l y i n d e p e n d e n t o f the e x u d a t i o n rate in the range o f 5-20 p l . h 1; a slight increase o f K + was observed at very low e x u d a t i o n rates (data n o t shown). W h e n the c o n c e n t r a t i o n o f sucrose was varied between 100 and 400 m M in the sievetube sap by i n c u b a t i o n o f the c o t y l e d o n s in media o f various c o n c e n t r a t i o n s o f sucrose, no obvious change in the K + c o n c e n t r a t i o n in the sieve-tube sap o c c u r r e d (Fig. 9). At c o n s t a n t sucrose c o n c e n t r a t i o n in the mediu m and varying c o n c e n t r a t i o n s o f K + the m o s t obvious effect o f K + was on the e x u d a t i o n rate. T h e r e was consistently an extremely low e x u d a t i o n rate at high K + c o n c e n t r a t i o n s (Fig. 10). T h e sucrose c o n c e n t r a t i o n in the sieve-tube sap was n o t
J. Kallarackal et al. : Sucrose transport into the sieve-tube of Ricinus
E
9
~80
9
~
9e
9
9
9
9
.~ 60~>
40-
~c 20~" o
o
16o 260 360 460 s6o 660 = sucrose in sieve tube sap (raM)
Fig. 9. Concentration of K + in the sieve-tuve sap of Ricinus seedlings at different concentrations of sucrose in the sieve-tuve sap. The cotyledons of seedlings, grown in vermiculite, were incubated in 5 mM potassium phosphate and various sucrose concentrations (0-200 mM) for 2 h. Then, exudation was started by cutting the hypocotyls and the exudate was analyzed for sucrose and K +
E
25 :=
-L~oosucrose concenfmfion
2
2O
300
,~ 20010 2
E
1002LJ
C3
o
io
do
K§
medium (raM)
do
N 0~
Fig. 10. Dependence of the exudation rate and the sucrose concentration in the sieve-tube sap of Ricinus seedlings on the K + concentration in the medium. The cotyledons of water-cultivated seedlings, from which the endosperm had been removed, were incubated in 50 mM sucrose for 2 h in media containing the indicated concentrations of K + (as phosphate). Then the hypocotyls were cut and exudation was followed
affected by K +, t h o u g h there a p p e a r e d to be a slight t e n d e n c y towards b o t h a higher flow rate and a higher sucrose c o n c e n t r a t i o n at approx. 1020 m M K +, which in c o m b i n a t i o n resulted in a 70% higher rate o f sucrose t r a n s l o c a t i o n at that K + c o n c e n t r a t i o n in the medium.
Estimation of the in-vivo rate of phloem transport and of the translocation speed. Cutting the h y p o c o t yl o f the Ricinus seedlings in order to obtain p h l o e m exudate r e m o v e d the sink tissues, namely the lower part o f the h y p o c o t y l and the r o o t system, and it is n o t k n o w n whether these tissues controlled p h l o e m t r a n s p o r t by limiting assimilate c o n s u m p t i o n . On the o t h e r hand, the slicing process could cause plugging o f some sieve tubes, thereby decreasing the rate o f solute flow far below that in the intact seedling. Therefore, a realistic estimate o f the in-vivo rate o f p h l o e m t r a n s p o r t
J. Kallarackal et al. : Sucrose transport into the sieve-tube of Ricinus Table 1. Dry weight of organs of Ricinus seedlings from days
4 to 6. The seedlings were grown in vermiculite for 4~6 d and ten seedlings were harvested per day, weighed, dried for 24 h at 80 ~ and weighed again. The SD was between 7 and 12% of the mean value. Respiration was measured with an oxygen electrode. The average seedling as used for the experiments was at the age of 5-6 d Dry weight (mg. (seedling)- 1)
Endosperm Cotyledons Hypocotyl Roots
day4
day5
283 32 22 21
246 43 33 34
Dry-weight change from day5 6 d a y 6 (mg.h ~ 9(seedling) - 1)
Respiration (mg sucrose .h 1(seed_ ling)-1)
88 90 109 46
-0.25 0.03} 0.23 0.26
--6.58 1.96 3.16} 0.5 3.66
had to be made to compare it with the rates of the exuding system. The net rate of phloem transport was calculated from the increase in dry weight of the hypocotyl and roots plus the respiratory loss of carbon by these organs (Table 1). The total dry-matter transfer through the phloem from the cotyledons to the hypocotyl amounted to 3.92 ragh - 1. (seedling)- 1. The rates of exudation of sievetube sap of seedlings, whose cotyledons were embedded in the endosperm varied between 20 and 40 gl-h 1, with the average at 30 pl.h -1. Since the sieve-tube sap contained 300 m M sucrose, 130 m M amino acids, 30 m M K + and probably 30 m M organic anions, a flow rate of 31 pl.h -1 would be required to account for the in-vivo net mass-transfer rate. We therefore conclude that the experimental system is not artificial in the sense that it causes entirely unnatural flow rates (Kallarackal and Milburn 1984). From the flow rate in vivo and during exudation the speed of phloem transport in the hypocotyl could be calculated. The Ricinus seedling has eight conducting bundles in the hypocotyl at the place of cutting, and these eight bundles together contain sieve tubes with a cross-sectional area of 0.030_+ 0.011 mm 2. A volume flow of 31 gl.h -1 means, if all sieve tubes are operating equally well, a flow speed of 1.03 m . h -1 or 12 r a m . r a i n - I ; the fastest exudation rate measured in a seedling was 47 pl. h-1, which corresponds to a flow speed of 1.6 m. h -1"
Discussion
There are several arguments, both theoretical and experimental, that the sap bleeding from the cut hypocotyl really stemmed from the sieve tubes. The
333
exudate consistently contained sucrose concentrations five- to ten-times higher than the bathing medium, and other solutes e.g. potassium and amino acids were also found to be concentrated. The p H of the sap was 7.55, a value close to the usually observed p H of sieve-tube sap. 3-Hydroxy-5,8,10pyrenetrisufonate, which is generally regarded as an apoplast marker (Dybing and Currier 1961), did not show up in the exuding sap when added to the medium, so that even minor contamination (2%) of the sieve-tube exudate with apoplast solutes can be ruled out within measuring accuracy. The exudation rate of cut seedlings, which still contained endosperm, was usually the same as that calculated from the growth increment for the net flow in the intact seedling. The mean speed of solvent flow, 1.03 m . h -1, is normal in the light of more recent literature (Minchin and Troughton 1980). One serious problem is the fact that the percentage of open sieve tubes after cutting is not controlled. It could be envisaged that where only a few sieve tubes are open the solutes mJ[ght be directed into these few conduits, resulting in a solute concentration larger than natural. :The comparison of volume flow rates and sucrose concentrations under otherwise identical experimental conditions showed that within the measured range there is no interdependence of these parameters. Results from seedlings with different flow rates are therefore comparable. It is unlikely that cutting the hypocotyl caused a surge or an increase in volume flow or an artificial change of solute concentration or solute composition. The first appearance of label in the exudate is noted within 10 rain after addition of label to the medium, but the uniform labelling of sieve-tube sap took up to 2 h; therefore, it appears that it is not the transfer of solutes from the medium to the sieve tubes but the uniform labelling of endogenous carbohydrate pools that is a slow process. The concentration of sucrose in the sieve-tube sap under natural conditions, with the endosperm attached is high, 250-300 raM, though not as high as measured with adult Ricinus (Smith and Milburn 1980 a-c), probably because water availability in the leaves of adult plants is always less than in the seedling. The concentration of sucrose in the sap and the exudation rate clearly behaved as in an osmotic system, as was obvious when an osmotic change in the medium occurred during exudation. Because the apoplastic concentration of solutes was probably identical to that of the medium it can safely be stated that at a particular apoplastic concentration the sieve-tube sap has a particular,
334 definite solute content. Thus, for the first time, a concentration dependence of sucrose loading into the phloem could be measured. At 100 m M sucrose in the medium the sucrose concentration in the sieve tubes has the same value as was determined using endosperm-attached cotyledons. The measured sucrose concentration at the endosperm-cotyledon interface was 70-90 m M (Kriedemann and Beevers 1967a; Komor 1977). An obvious difference between our seedling system and the few known systems with adult plants is the ease with which the sucrose concentration in the sieve-tube sap can be varied by altering the supply of external sucrose. For instance in adult plants the day-night rhythm showed little variation (Smith and Milburn 1980a), whereas in the seedlings the sucrose concentration in sievetube sap decreased to one-fourth within 2 h when internal, stored carbohydrates were the only source. For many years, the effect of K § on sucrose loading has been a common topic in the literature (Mengel and H/ider 1977; Lang 1979). Usually the correlation between sucrose and K + concentration was determined, since it was not really possible to apply defined concentrations of K § to the apoplast and to monitor simultaneously the sucrose transport into the sieve tubes. The Ricinus seedling system has allowed this experiment to be carried out for the first time, and has demonstrated that there is a pronounced effect of K § application on the K + content of sieve-tube sap (data not shown) but definitely not on the sucrose content. If there is a promotion of phloem loading by K § then either it occurs prior to an apoplastic loading step, for instance at the exit of sucrose from the mesophyll, or the very low concentration of K § found in situ is already sufficient for maximal activity. What are the advantages and disadvantages of the exuding Ricinus seedling system compared with other known systems for the analysis of sieve-tube sap (Ziegler 1975)? i) The Ricinus seedling usually exudes sieve-tube sap only for 2 3 h, but that period can be prolonged by further slicing. Much longer periods have been reported for incisions of adult plants or for the cut stylets of aphids, (though very short exudation periods can also occur with the latter), ii) The sample volumes are smaller than from cut adult Ricinus, but are by far larger than from aphid stylets. Compared with the latter, chemical analysis is much easier and evaporation is a much smaller problem, iii) The percentage of open sieve tubes after cutting is unknown, and in this respect the situation is similar to that of incised adult plants (and to aphid experi-
J. Kallarackalet al. : Sucrose transport into the sieve-tubeof Ricinus ments, too, where partial plugging is possible). Therefore it is not possible to draw conclusions about mass-transfer rates in a single, isolated experimental situation, iv) Cutting the hypocotyl of the seedling does not increase the flow rate of sievetube sap. Therefore, artificial effects caused by a change in the water flow do not have to be taken into account. In adult plants, incised or infested with aphids, this is a serious problem, v) Both the exuding Ricinus seedling and the incision of adult plants allow the determination of the real concentration of compounds in the sieve-tube sap. This is in contrast to the leakage caused by ethylenediaminetetraacetic acid (King and Zeevart 1974), and also to the aphid method, where considerable evaporation has to be envisaged because of the minute exudate volumes, vi) The exuding Ricinus seedling is the only system where the uptake o f ' particular compounds by the leaves, either natural compounds or xenobiotics, and the composition of sieve-tube sap can be monitored simultaneously. Since the compounds can be offered at defined concentrations to the apoplast, a quantitative analysis of the concentration dependence of phloem loading for that particular compound is possible. Future experiments with the Ricinus seedling may help to determine which factors regulate phloem loading, which solutes besides sucrose can be loaded into the sieve tubes, and which concentrations of particular solutes are achieved in the sieve tubes when the concentration and conditions in the apoplast are defined. During revision of this manuscript, a report appeared describing experiments involving exudation from the cut hypocotyl of Ricinus seedlings (Vreugdenhil and Koot-Gronsveld 1988). Only seedlings with the endosperm attached were used, and their results were essentiallythe same as those reported by us in the first section of the Results. The experimentalhelp given by Klaus Wassermannand Walter K6ckenberger, and the financial funding by Deutsche Forschungsgemeinschaftand by A.v. Humboldt-Stiftung(to Jose Kallarackal) are gratefullyacknowledged References Daie, J. (1987) Sucrose uptake in isolated phloem segments of celery is a single saturable transport system. Planta 171, 474482 Dybing, D.C., Currier, H.B. (1961) Foliar penetration by chemicals. Plant Physiol.36, 169 174 Fisher, D.B. (1981) Measurement of the sieve tube membrane potential. Plant Physiol.67, 845 849 Hall, S.M., Baker, D.A., Milburn, J.A. (1971) Phloemtransport of 14C-labelledassimilatesin Rieinus. Planta 100, 200-207 Kallarackal, J., Milburn, J.A. (1984) Specificmass transfer and sink-controlled phloem translocation in castor bean. Aust. J. Plant Physiol. 11,483~490
J. Kallarackal et al. : Sucrose transport into the sieve-tube of Ricinus King, R.W., Zeevart, J.A.D. (1974) Enhancement of phloem exudation from cut petioles by chelating agents. PIant Physiol. 53, 96-103 Komor, E. (1982) Transport of sugar. In: Encyclopedia of ptant physiology, N.S., vol. :I3A Plant Carbohydrates I. IntracelIular Carbohydrates, pp. 635-676, Loewus, F.A., Tanner, W., eds., Springer, Berlin Heidelberg Komor, E. (1977) Sucrose uptake by cotyledons of Ricinus communis L. Characteristics, mechanism and regulation. Planta 137, 119-131 Kriedemann, P., Beevers, H. (1967 a) Sugar uptake and transtocation in the castor bean seedling. I. Characteristics of transfer in intact and excised seedlings. Plant Physiol. 42, 161-173 Kriedemann, P., Beevers, H. (1967 b) Sugar uptake and translocation in the castor bean seedling. II. Sugar transformations during uptake. Plant Physiol. 42, 17z1~180 Kursanov, A.L. (1984) Assimilate transport in plants. Elsevier, Amsterdam Lang, A. (1979) A relay mechanism for phloem translocation. Ann. Bot. 44, 141-I45 Mengel, K., H/ider, H.E. (1977) Effect of potassium suppIy on the rate of phloem sap exudation and the composition of phloem sap of Ricinus communis. Plant Physiol. 59, 282284 Minchin, P.E., Troughton, J.H. (1980) Quantitative interpretation of phloem translocation data. Annu. Rev. Plant Physiol. 31, 191-215
335 Schobert, C., Komor, E. (1987) Amino acid uptake by Ricinus communis roots: characterization and physiologicaI significance. Plant Cell Environ. 10, 493 500 Smitb, J.A.C., Milburn, J.A. (1980a) Osmoregulation and the control of phloem-sap composition in Ricinus communis L. Planta 148, 28 34 Smith, J.A.C., Milburn, J.A. (1980b) Phloem transport, solute flux and the kinetics of sap exudation in Ricinus communis L. Planta 148, 35-41 Smith, J.A.C., Milburn, J.A. (1980c) Phloem turgot and the regulation of sucrose loading in Ricinus communis L. Planta 148, 42~48 Sovonick, S.A., Geiger, D.R., Fellows, R.J. (1974) Evidence for active phloem loading in the minor veins Of sugar beet. Plant Physiol. 54, 886-891 Vreugdenhil, D., Koot-Gronsveld, E.A.M. (1988) Characterization of phloem exudation from castor-bean cotyledons. Planta 174, 38(~384 Yemm, E.W., Willis, A.J. (1954) The estimation of carbohydrates in plant extracts by anthrone. Biochem. J. 57, 508 514 Ziegler, H. (1975) Nature of transported substances. In: Encyclopedia of plant physiol., N.S., vol. 1 : Transpbrt in plants I. Phloem transport pp. 59-100 Zimmerman~ M.H. Milburn, J.D. eds., Springer, Berhn Heidelberg New York Received 9 March: accepted 5 October 1988
