Planta
Planta (1982)155:82 88
9 Springer-Verlag 1982
Phloem transport of sulfur in Ricinus U. Bonas*, K. Schmitz, H. Rennenberg, and L. B e r g m a n n * * Botanisches Institut der Universitgt, Gyrhofstrasse 15, D-5000 K61n 41, Federal Republic of Germany
Abstract. Mature leaves of R i c i n u s c o m m u n i s
fed with 35SO2- in the light export labeled sulfate and reduced sulfur c o m p o u n d s by phloem transport. Only 1 2% of the absorbed radiosulfur is exported to the stem within 2-3 h, roughly 12% of 3sS recovered was in reduced form. The composition of phloem translocate moving down the stem t o w a r d the root was determined f r o m phloem exudate: 2 0 - 4 0 % o f the 35S m o v e d in the form o f organic sulfur c o m p o u n d s , however, the bulk o f sulfur was transported as inorganic sulfate. The most i m p o r t a n t organic sulfur c o m p o u n d translocated was glutathione, carrying a b o u t 70% o f the label present in the organic fraction. I n addition, methionine and cysteine were involved in p h l o e m sulfur transport and accounted for roughly 10%. Primarily, the reduced forms of both, glutathione and cysteine are present in the sieve tubes. Key words: P h l o e m exudate - P h l o e m transport (sulfur) - R i c i n u s - Sulfur - Glutathione.
To make sulfate available for reduction, it is distributed with the transpiration stream and m a y either be reduced at the sites of highest d e m a n d in adequate quantities, or source-sink relationships for reduced sulfur m a y d e m a n d a transport of these c o m p o u n d s . Previously published results (Rennenberg et al. 1979) have shown that tobacco plants export organic sulfur c o m p o u n d s f r o m mature leaves and translocate them t o w a r d sink regions, i.e., the shoot apex, i m m a t u r e leaves, and the r o o t system. This transport occurred at least partially in the phloem. Separation and identification of the organic sulfur translocated revealed that glutathione is the major c o m p o n e n t of the translocate. We report here on transport of sulfate and reduced sulfur c o m p o u n d s in R i c i n u s , with emphasis on the localization o f the translocation p a t h w a y and a m o r e detailed analysis of the translocate f r o m phloem exudate. Material and methods
Introduction
C o r m o p h y t e s cover their sulfur supply nearly exclusively by sulfate uptake f r o m the soil. Since mainly reduced sulfur is b r o u g h t into organic binding, oxidized sulfur has to be reduced before it can be incorporated into c a r b o n skeletons. T h o u g h roots are able to reduce sulfur to some extent (Pate 1965; Ellis 1969), by far m o r e reduction takes place in the leaves, most likely in the chloroplasts (Schwenn and Trebst 1976; Schmidt 1979; A n d e r s o n 1980). * Present address: Max-Planck-Institut ffir Zfichtungsforschung,
D-5000 K61n 30 Federal Republic of Germany ** To whom reprint requests should be sent Abbreviations: CySH=cysteine; GSH=glutathione; GSSG=glutathione disulfide; NEM =N-ethylmaleimide CyS-SCy=cystine
0032-0935/82/0155/0082/$01.40
Plant material and experimental conditions. Ricinus communis L. var. gibsonii Nichols. were grown as pot plants in a heated glass-
house (18-20~ C at night, up to 30~ C at daytime) under natural light conditions, supplemented by additional illumination (Osram L Flnora 65 W, 12 gmol photons m-Zs -1, 16 h photoperiod). Two days before an experiment was started, eight- to eleven-week-old, vigorously growing plants were brought to the laboratory and adapted to the experimental conditions (200-250 gmol photons m-Zs 1, 16 h photoperiod, 25 27~ C). All but the very young leaves at the shoot apex and one mature leaf halfway along the stem were removed. It was observed that the general distribution pattern for leaf fed 3sS was the same in intact plants and in plants having all but one mature leaf detached (experiments not presented). In the present experiments, the plants were prepared in such a way as to reduce the complexity of the transport system. Experiments were started, at the earliest, 48 h after the leaves were cut ; a serious impact of wounding on long-distance transport was not observed. Phloem exudate was collected with graduated glass capillaries from diagonal incisions across a bark strip, which remained intact
U. Bonas et al. : Phloem transport of sulfur
83
after the internode of the fed leaf or the next internode below had been partially girdled at least one day before an experiment was started (c.f. Smith and Milburn 1980). 35SO2 (185 MBq/mgS) was supplied to the leaf using the flap-feeding technic introduced by Biddulph (1941).
Analysis of tissue samples. At the end of an experiment, the stem and petiole were rapidly dissected into 40 m m pieces, frozen and lyophilized. An aliquot of powdered dry weight of each sample was deproteinized (heat denaturation), and 3SS-labeled c o m p o u n d s were extracted with distilled water. Glutathione (GSH) was oxidized to glutathione disulfide (GSSG) according to Jones and Carnegie (1971). The combined water extracts containing GSSG and other 35S-labeled c o m p o u n d s were frozen and lyophilized. The dry residue was resuspended in distilled water and the radioactivity determined by liquid scintillation counting (LSC). Sulfate was precipitated with BaClz at 4 ~ C followed by repeated coprecipitation with N % S O 4. TSe radioactivity of the organic sulfur c o m p o u n d s in the supernatant was measured by LSC. [35S]sulfate activity was calculated. Prior to a chromatographic separation of the organic sulfur compounds, samples had to be further purified by an ion exchange column (Dowex 50 W x 4, 50-100 mesh). 3~S-compounds were washed from the column with 10 N NH3. This effluent was evaporated to dryness, resuspended in 0.2-1 ml distilled water, and further separated using thin-layer chromatography or thinlayer electrophoresis as outlined below. 35S-labeled protein and protein-bound sulfur c o m p o u n d s were determined after sulfate and low molecular weight organic sulfur c o m p o u n d s had been removed: Sulfur c o m p o u n d s b o u n d to protein via disulfide bridges were washed off with borate-buffered dithioerythritol (0.1%, pH 8.6). The residue was treated with 1.5% N a O H to dissolve the protein. Radioactivity of both, protein-bound c o m p o u n d s and protein, were determined by LSC.
Analysis of blade samples. Tissue samples of the fed blade (lamina, veins and petiole), up to 650 mg dry weight, were homogenized at 4 ~ C with 5 ml 80% ethanol and 25 m M N-ethylmaleimide (NEM). During a reaction time of 60 min at 25 ~ C, the substances
with sulfhydryl groups formed stable alkyl derivatives (Gregory 1955). The homogenate was centrifuged for 20rain at 10,000g. The sediment was extracted with 25 m M N E M in 80% ethanol followed by two water extractions, the first at I00 ~ C, and the second at 20 ~ C. The combined extracts were evaporated to l - 2 ml. Further qualitative and quantitative analysis was the same as described for samples of the stem and analysis of exudate, respectively.
Analysis of exudate. Samples of exudate were collected in 10-rain batches for several hours, beginning 20-30 min after [35S]sulfate feeding was started. The volume of exudate was determined and immediately mixed with a three-fold volume of 25 m M N E M in 80% ethanol. After a 60-min incubation at 25 ~ C and subsequent centrifugation, radioactivity of the supernatant containing [3 SS]sul_ fate and 3SS-labeled organic c o m p o u n d s was measured. [3SS]sulfate was separated from other labeled c o m p o u n d s by thin-layer electrophoresis on cellulose (cellulose: M N 300, 0.1 m m thick; verona1 buffer: 5,5-dietbylbarbiturate sodium salt, Serva, 0.075 M, p H 8.6; 600 V; 25 rain). The same system was applied for 90 min to separate the fraction of low molecular weight, 3SS-labeled compounds. Alternatively, these c o m p o u n d s were separated by thin-layer chromatography on cellulose plates ( M N 300), one dimensionaly. Solvents: n-butanol:acetic a c i d : H 2 0 (60:15:25) (by vol.) or two runs in n-propanol: formic acid : H 2 0 (65 : 1 : 34) (by vol.). The radioactivity of separated c o m p o u n d s was recorded with a thin-layer scanner, or the adsorbent containing the substance was scraped from the plate and measured by LSC. Substances were identified by cochromatography and rechromatography with authentic compounds.
Results
Movement o f 35 S in castor bean plants. When [3 s S]sulfate was supplied to a mature leaf by flap-feeding via two major veins for 2 h, 35S was exported toward the shoot apex as well as the root. An example is given in Table 1. Reduction of sulfate, presumably
Table 1. Distribution of 35S in stem and roots of Ricinus communis after a 2 h translocation periode. 35SO 2- was continuously supplied to a mature leaf. The topography of sampling a n d the distribution of 35S-labeled organic c o m p o u n d s and sulfate are shown Dry weight
kBq c m - 1 stem Sulfate
175 198 246 225 333 333 419 397 524 522 642 685 926 706 626 644 6,480
14.38 3.95 1.8i 1.60 2.78 3.70 5.10 3.94 3.97 2.44 t .98 1.81 1.77 2.03 i .88 3.27 56.70
Reduced soluble sulfur 7.04 1.42 1.27 1.00 1.64 2.29 3.19 2.04 2.31 1.63 1.33 1.12 1.00 1.02 0.78 0.63 10.23
Reduced sulfur % of total soluble 35S
kBq.100mg dryweight-1
Sample topography
Sulfate
Reduced solubIe sulfur
Protein
32.9 26.4 41.2 38.5 37.1 38.2 38.5 34.1 36.8 40.0 40.2 38.2 36.1 33.4 29.3 16.1 15.3
8.22 7.98 3.03 2.85 3.34 4.44 4.87 3.97 2.95 1.87 1.30 1.05 0.96 i. 15 1.20 2.44 0.87
4.03 2.87 2.12 1.77 1.97 2.75 3.05 2.05 1.72 1.25 0.88 0.65 0.54 0.58 0.49 0.47 0.16
0.83 0.23 0.11 0.11 0.14 0.18 0.17 0.12 0.10 0.08 0.06 0.05 0.04 0.04 0.03 0,03 0.01
~3 II
84 Q)
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84
U. Bonas et al. : Phloem transport of sulfur
Table 2. Budget of the uptake, reduction, and distribution of 35S in a Ricinus plant during a 2 h period of continuous feeding of 35SO~- to a mature leaf. All other leaves were removed 48 h before the experiment was started. Radioactivity is given in kBq- 102 Sample
Water soluble
Non-water soluble
Total 3sS
% of 35Suptake
Sulfate
Reduced sulfur
Protein
Protein b o u n d sulfur compounds
527.3 1.138
51.02 0,279
6.601 0.026
8,767 0.012
593.688 1.455
98.81 0.24
Export acropetally stem
1.759 0.556
1.065 0,306
0.200 0.022
0.094 0.022
3.118
0.52
shoot apex with youngest leaves
1,203
0.759
0. I78
0.072
Export basipetally stem root
1.737 0.858 0.879
0,720 0,516 0,204
0.047 0.033 0.014
0.100 0,050 0.050
2.604
0.43
600.865
100.00
Fed leaf lamina petiole
Whole plant
585.018
in the chloroplasts, provided appreciable radioactivity confined to low molecular weight substances which moved out of the source leaf. Besides, [35S]sulfate was translocated and distributed acro- and basipetally. There was usually a gradient of decreasing radioactivity from the source leaf to the shoot apex as well as to the root. The percentage of reduced sulfur ranged between 15 and 40% of the total water soluble radioactivity. This relation was relatively constant in the stem (38-40%), but smaller in the shoot apex (33%) and the root system (33-15%). The root and shoot apex contained more [35S]sulfate than the stem pieces. These observations basically remain the same whether they are related to the length of transport pathway or the dry weight (Table 1). It appears, however, that the shoot apex during the 2-h experiment slightly accumulated sulfate and reduced sulfur compounds. The total uptake and distribution of 35S in the plant is shown in Table 2. During the 2-h feeding period, which was the total experimental time, 1.43 ml [35S]sulfate soiution with a radioactivity of 63.9 MBq was absorbed. Approximately 99% of this radioactivity remained in the fed leaf whereas only 1% was exported to the stem. Roughly the same amount of radioactivity was directed toward the shoot apex and the root. Nearly 12% of the absorbed [35S]sulfate was reduced within the 2 h and detected in low molecular weight organic substances, protein, and proteinattached sulfur compounds. Analysis of the water-soluble 35S-compounds of stem segments by thin-layer chromatography revealed that apical to the fed leaf, 77% of the radioactivity in this fraction was confined to glutathione, 5.6% to cysteine, 4% to methionine; below the source leaf
15.847
76% accounted to glutathione, 8.8% to cysteine, and 3.6% to methionine (average of 7 separations).
Distribution of radiosulfur in the leaf. To obtain further information about the loading of organic and inorganic sulfur compounds into the long-distance transport pathway, a leaf was flap-fed with [35S]sulfate via the midvein for 60 min while, at the same time, phloem exudate was collected from the internode below. Thereafter, samples of the lamina, the veins, and the petiole were analyzed for the distribution of [35S]sulfate and reduced radiosulfur. Lamina samples and the veins contained 4-5,4% reduced 35S, whereas the petiole exhibited up to 21% reduced asscompounds. There was an increasing gradient of radioactivity along the petiole, indicating that a lower percentage of reduced sulfur entered the petiole (17 %) and that a higher percentage was loaded into the long-distance transport system of the stem (21%). The composition of the organic sulfur compounds in the leaf is shown in Table 3. Following the path of these compounds from the mesophylI to the veins, along the veins to the petiole, and along the petiole to the transport system of the stem, it may be noted that methionine comprises a much higher radioactivity in samples of the mesophyll and the veins than in the petiole, whereas cysteine shows just the opposite. The reduced form of glutathione also shows a higher label of radioactivity in the petiole than in the mesophyll and the veins, whereas oxidized glutathione carries higher 35S-labeling in the mesophyll and vein samples than in the petiole. The experimental data presented so far indicate that sulfate as well as organic sulfur compounds are phloem mobile. At least export through the petiole
U. Bonas et al. : Phloem transport of sulfur
85
Table 3. Distribution of 35S a m o n g organic sulfur c o m p o u n d s in samples of lamina, veins, and petiole, separated by thin-layer electrophoresis (Met methionine; C y S H = cysteine; G S H = g l u t a t h i o n e ; G S S G = g l u t a t h i o n e disulfide; CyS-SCys=cystine Sample
% of reduced sulfur c o m p o u n d s
Lamina Veins
Met
CySH
13.0 11.5
6.4 11.1
3.6 5.3
12.1 7.0
Petiole upper half lower half
CyS-SCy
GSH
GSSG
Unidentified
5.7 5.0
28.9 31.2
28.1 25.7
17,9 15.5
13.1 15.3
39.0 39.7
20.8 21.5
11,4 I 1.2
and along the stem toward the root point to a transport in the phloem, but translocation up toward the shoot apex could involve phloem as well as xylem transport.
O
~
4 "7
•
/
g-~2
O
/~
0 ~
e
i
40
A__A~A~ e
~
r
60
i
80
9 F
e
e
i
i
e
e
i
e
r
D
100 120 140 160 180 Time (min)
Fig. 1. Releaseof 35S-labeledorganic compounds (A) and [35S]sulfate (o) with phloem exudate. A mature leaf was continuously fed with 35SO4Z-. Collection of phloem exudate started after 35 min at a site 34 cm from the source leaf
Detection of translocated 35S-labeled compounds in phloem exudate. Analysis of translocated compounds shown above is based on extracts of stem segments and blade samples; however, the distribution of 35S obtained in this way must not necessarily be identical to the real composition of the translocate. A better localization of sulfur transport and better characterization of sieve tube content was expected from phloem exudate analysis. An experiment in which a mature leaf of a Ricinus plant was continuously fed with [3SS]sulfate and collection of phloem exudate started 35 rain later is presented in Fig. 1. The collecting site was 34 cm below the source leaf. 35S was already detected in the first
Table 4. Exudate analysis of Ricinus communis after continuous feeding with 35SO~- to mature leaf. Exudate collection began 25 rain after the experiment was started Experiment time (min)
Volume (~d)
25 35 35- 45 45 55 55- 65 65- 75 75- 85 85 95 95-105 105 115 115-125 125 135 135-145 145 155
42 70 85 123 60 90 110 125 120 120 145 161 135 151 i28 115
155-165
165 175 175 185
kBq. 10 gi -I 35SO2-
0.01 0.59 1.16 1.52 1.83 2.23 2.52 2.03 1.75 1.72 2.15 2.08 2.26 2.19 1.71 2.31
Reduced sulfur
Reduced sulfur (% of solubles)
0 0.03 0.18 0.38 0.58 1.19 0.84 0.96 1.06 1.14 0.82 0.98 0.94 0.68 0.78 0.62
0 4.8 13.4 20.0 24.1 34.8 25.0 32.1 37.7 39.9 27.6 32.0 29.4 23.7 31.3 21.2
% of 3sS in reduced sulfur c o m p o u n d s Met
CySH
CyS-SCy
GSH
GSSG
Unidentified
17.0 8.7 7.0 4.2 3.7 3.4 4.0 3.7 3.7 3.6 3.3 2.8 3.5 3.2 2.8
22.0 18.9 11.0 9.8 7.2 6.7 6.9 6.4 4.8 5.6 4.9 3.5 4.0 3.6 3.1
9.0 6.7 3.0 2.0 1.8 1.9 2.3 1.7 2,2 1.8 1.9 1.8 2.9 2.0 2.0
27.6 42.3 45.8 50.1 45.6 48.4 43.5 45.8 46.1 44.2 43.1 42.0 35.6 37.6 34.9
7.7 13.9 22.2 20.8 24.4 24.0 26.3 24.7 25.2 25.2 26.8 29.1 29.0 29.6 31.1
16.7 9.5 11.0 13.1 17.3 15.6 17.0 17.7 18.0 19.6 20.0 20.8 25.0 24.0 26.1
86
U. Bonas et al. : Phloem transport of sulfur ,o-. o
350 300
o
\
250 "7
o
o
200
0 x
O" m
150
o o
100 50
Af
o
30
A/
l
60
9'0
189
150 180 Time(min)
Fig. 2. Pulse feeding experiment. 35SO2- was fed for 3 min followed by a chase periode of 180 min. The profiles of 35S-labelled organic compounds (A) and sulfate (o) released with phloem exudate are shown. The collecting site was 34 cm below the labeled leaf 10-rain batch of exudate, indicating a transport velocity of at least 60 cm h - t . Radioactivity steadily increased with time for 180 min (Table 4). There was a steep increase of [35S]sulfate which finally reached a plateau after 120 min, whereas labeled organic sulfur compounds steadily increased in radioactivity. The rate of exudation varied considerably between 4 and 18.5 gl min 1; within 3 h, almost 1.8 ml exudate were collected containing 1.75% ass absorbed by the leaf. Pulse feeding of [3SS]sulfate for 3 min with a subsequent chase periode of 180 min feeding cold salt solution (Murashige and Skoog 1962), resulted in a release of radioactivity detected 40 min later in the exudate collected 34 cm below (Fig. 2). While reduced, labeled sulfur compounds in the exudate steadily increased with time, the profile for [3SS]sulfate showed a sharp peak, reaching a maximum between 60 and 70 min after the pulse was given. A representative record of 3sS release based on phloem exudate analysis from a plant that was continuously fed is presented in Table 3. Exudate was collected for 185 rain and 10-min batches were analyzed. The exudation rate again varied between 4.2 and 16.1 lal min-1. It was low in the beginning but gradually increased to a level of 13 gl min-1 within the first 2 h of the experiment and subsequently remained constant. [3SS]sulfate as well as reduced sulfur compounds were detected in the phloem sap with both fi'actions exhibiting saturation kinetics. Radioactivity
of the reduced sulfur fraction ranged between 25 and 40% of soluble 3sS. Separation of organic sulfur compounds of phloem exudate by thin-layer electrophoresis revealed the following, clearly identified, radioactively labeled compounds: methionine, cysteine, cystine, reduced, and oxidized glutathione. The highest percentage of 35S was confined to glutathione. The reduced form (GSH) revealed on an average 4 3 % , the oxidized form (GSSG) 26% of asS-labeled soluble organic compounds. In other experiments not presented here, up to 85% of the radioactivity was confined to glutathione. The percentage of 35S in methi0nine was 3.5%, in cysteine 6%, and in cystine 2%. From 60 min on, fluctuation of the percentage distribution among these compounds was minor. A rather high percentage of radioactivity on the thin-layer plates was not identified, 3-4% of the non-identified compound(s) are due to substance(s) which did not chromatograph, but remained at the origin. Discussion
The source of sulfur for the synthesis of many essential sulfur compounds in cormophytic plants is inorganic sulfate taken up through the root. Sulfate is rapidly distributed with the transpiration stream throughout the plant (Biddulph et al. 1958). Pulse feeding experiments with bean plants revealed that 35S applied to the root first accumulated in mature leaves. Twelve - twenty-four hours later, such leaves had lost their radioactivity to the young leaves at the stem apex, indicating an export of 3sS (Biddulph et al. 1958). When 35S was fed to a bean leaf using the flap-feeding technic (Biddulph 1941), radioactivity moved out of the fed leaf and down the stem, mainly through the phloem, as shown by histoautoradiography (Biddulph 1956). Phloem mobility of sulfur was also reported by Weigl and Ziegler (1962), Peel (1970), Rennenberg et al. (1979), Garsed and Mochrie (1980) and is shown here for Ricinus. Movement down the stem usually excludes movement in the xylem, whereas upward movement might either be localized in the xylem, in the phloem, or in both since a lateral exchange cannot be excluded. One might argue that sulfur, introduced by a leaf flap and assimilated in the mesophyll, is passively swept out of the leaf with the assimilate stream whenever sulfur compounds enter the sieve tubes. The analysis of 3sS in the lamina and along the conducting pathway revealed, however, that the relative amount of reduced organic sulfur compounds in the lamina was much less (9%) than in the petiole (20%) and in the stem (36%), which indicates that preferably organic sulfur compounds may be loaded into the sieve elements (c.f. Table 2). Though a high accumula-
U. Bonas et al. : Phloem transport of sulfur
tion capacity of phloem, especially of sieve elements, for ions (phosphate, sulfate) has previously been reported (Bieleski 1966), the present experiments indicate a selective permeability, favoring the entrance of reduced sulfur compounds over sulfate. It is assumed that the distribution of ass follows source-sink relationships for reduced sulfur compounds and sulfate. The directionality is probably not determined solely by concentration gradients for sulfur compounds, but by an overall concentration gradient for ions, carbon, nitrogen-, and sulfur assimilates. Transport of ass in the phloem was clearly demonstrated here by phloem sap analysis. Release of phloem exudate from bark incisions of Ricinus was reported before, and circumstantial evidence was presented that phloem exudate represents sieve tube content (Milburn 1971 ; Hall et al. 1971 ; Hall and Baker 1972; Smith and Milburn 1980). Under this assumption the chemical composition of the sap should reflect the translocate. The analysis of phloem exudate of Ricinus revealed that the main radioactivity was confined to inorganic sulfate. Sulfate is known to be an anionic component of Ricinus phloem exudate though its concentration is rather low (0,024-0,048 mg m l - 1) (Hall and Baker 1972). Sulfate is considered to play a minor role in phloem transport of sulfur as compared to the sulfur movement in the organic form (Ziegler 1975). This is in contradiction to results presented here, demonstrating that only 30-40% of ass in the translocate is in organic binding. The conclusive answer to this question can only be obtained by a determination of the specific radioactivity; the absolute quantities of sulfate and sulfur amino acids, however, were not measured. It is only known that methionine is present in trace amounts in sieve tube sap (Hall and Baker 1972). The percentage distribution of radiosulfur among sulfate and organic sulfur compounds was nearly the same in stem segments and phloem exudate, indicating that the ass-labeled translocate probably did not leave the long distance transport channel or was not further metabolized after it might have left the sieve tubes (c.f. Tables 2, 4). The main labeled organic compound in phloem sap of Ricinus is glutathione. The reduced form (GSH) revealed a much higher radioactive labeling than the oxidized form (GSSG) (Table 4). Provided that the specific activity of glutathione is more or less constant after 60 min, the absolute amount of GSSG is not 50% that of GSH, as indicated by Table 4, but only one quart, since one molecule GSSG carries two sulfur atoms. It seems that glutathione is translocated in the reduced form. This was further confirmed by electrophoresis of exu-
87
date immediately treated with N E M to prevent oxidation. Therefore, the oxidized form of glutathione (GSSG) ranged between 25 and 35% total glutathione-sulfur, the percentage of cystine (CyS-SCy) was 30-40% total cysteine-sulfur, indicating that the reduced substances are the favored transport forms. The present experiments support the idea that green cells of higher plants are able to reduce more sulfate than necessary for their own needs and to incorporate this surplus into glutathione that is translocated to places of high demand, i.e., the apex and the root system (Rennenberg et al. 1979). Though the experiments were not designed to measure exact translocation velocities, rough figures can be given even though translocation velocity is variable and dependent on a number of exogenous and endogenous factors. Velocity of asS-export of leaf-fed sulfate was 30 cm h -1 in " i n t a c t " plants (Table 1) and at least 58 cm h -1 in exuding stems (Fig. 1, 2). Hall et al. (1971) reported a velocity of > 100 cm h 1 for the translocation of ~4C-labeled assimilates in exuding Ricinus plants and 80-84 cm h - ~ in intact plants. Translocation velocities > 63 cm h - 1 were derived by Smith and Milburn (1980) from measurements of mass transfer. It appears that transport velocity is higher in exuding plants as compared to intact plant systems. This apparently has an impact on translocation rate. Although we have not determined the specific mass transfer, our results indicate that sulfur transport might be more efficient in the exuding stem than in intact plants. The results presented in Table 1 and Table 2 show that in an intact Ricinus plant roughly 1% of total ass absorbed moved out of the fed leaf and along the stem toward the root and the apex. In the exuding plant (Fig. 1), 1.7% were collected with the phloem sap, indicating a higher export if movement of ass toward the shoot apex is taken into account. These results may be explained by the assumption that bark incisions create a high, artificial sink, attracting most of the translocate. The specific mass transfer of the enhanced phloem transport in exuding plants must have been quite high, since the conducting cross-sectional area of phloem was reduced to approximately 30% by partial girdling of the stem. Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.
References Anderson, J.W. (1980) Assimilation of inorganic sulfate into cysteine. In: The biochemistry of plants, vol. 5, pp. 203 223, Miflin, B.J., ed. Academic Press, New York Biddulph, O. (i941) Diurnal migration of injected radiophosphorus from bean leaves. Am. J. Bot. 28, 348 352
88 Biddulph, S.F. (1956) Visual indications of S 35 and p32 translocation in the phloem. Am. J. Bot. 43, 143-148 Biddulph, O., Biddulph, S.F., Cory, R., Koontz, H. (1958) Circulation patterns for p~2 S3S and Ca 45 in the bean plant. Plant Physiol. 33, 293-300 Bieleski, R.L. (1966) Sites of accumulation in excised phloem and vascular tissues. Plant Physiol. 41, 455 466 Ellis, R.J. (1969) Sulfate activation in higher plants. Planta 88, 3442 Garsed, S.G., Mochrie, A. (1980) Translocation of sulphite in Vicia f a b a L. New Phytol. 84, 421 428 Gregory (1955) The stability of N-ethylmaleimide and its reaction with sulfhydryl groups. J. Am. Chem. Soc. 77, 3922-3923 Hall, S.M., Baker, D.A. (1972) The chemical composition of Ricinus phloem exudate. Planta 106, 131 140 Hall, S.M., Baker, D.A., Milburn, J.A. (1971) Phloem transport of 14C-labelled assimilates in Ricinus. Planta 100, 200-207 Jones, J.K., Carnegie, P.R. (1971) Binding of oxidized glutathione to dough proteins and a new explanation, involving thioldisulfide exchange, of the physical properties of dough. J. Sci. Food. Agric. 22, 358-364 Milburn, J.A. (1971) An analysis of the response in phloem exudation on application of massage to Ricinus. Planta 100, 143-154 Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. lfi, 473 497
U. Bonas et al. : Phloem transport of sulfur Pate, J.S. (1965) Roots as organs of assimilation of sulfate. Science 149, 547 548 Peel, A.J. (1970) Further evidence for the relative immobility of water in sieve tubes of willow. Physiol. Plant. 23, 667 672 Rennenberg, K., Schmitz, K., Bergmann, L. (1979) Long-distance transport of sulfur in Nicotiana tabacum. Planta 147, 57-62 Schmidt, A. (1979) Photosynthetic assimilation of sulfur compounds. In: Encyclopedia of plant physiology, N.S., vol. 6, pp. 481496, Gibbs, M., Latzko, E., eds. Springer, Berlin Heidelberg New York Schwenn, J.D., Trebst, A. (1976) Photosynthetic sulfate reduction by chloroplasts. In : The intact chloroplast, pp. 315 334, Barber, J., ed. Elsevier, Amsterdam New York Oxford Smith, J.A.C., Milburn, J.A. (1980) Phloem transport, solute flux and the kinetics of sap exudation in Ricinus communis L. Planta 148, 35-41 Weigl, J., Ziegler, H. (1962) Die rfiumliche Verteilung von 3sS und die Art der markierten Verbindungen in Spinatblfittern nach Begasung mit 35SO2. Planta 58, 435 447 Ziegler, H. (1975) Nature of substances in the phloem. In: Encyclopedia of plant physiology, N.S., vol. 1, pp. 59-100, Zimmermann, M.H., Milburn, J.A., eds. Springer, Berlin Heidelberg New York
Received 6 February; accepted 4 March 1982
Planta (1982)155:82 88
9 Springer-Verlag 1982
Phloem transport of sulfur in Ricinus U. Bonas*, K. Schmitz, H. Rennenberg, and L. B e r g m a n n * * Botanisches Institut der Universitgt, Gyrhofstrasse 15, D-5000 K61n 41, Federal Republic of Germany
Abstract. Mature leaves of R i c i n u s c o m m u n i s
fed with 35SO2- in the light export labeled sulfate and reduced sulfur c o m p o u n d s by phloem transport. Only 1 2% of the absorbed radiosulfur is exported to the stem within 2-3 h, roughly 12% of 3sS recovered was in reduced form. The composition of phloem translocate moving down the stem t o w a r d the root was determined f r o m phloem exudate: 2 0 - 4 0 % o f the 35S m o v e d in the form o f organic sulfur c o m p o u n d s , however, the bulk o f sulfur was transported as inorganic sulfate. The most i m p o r t a n t organic sulfur c o m p o u n d translocated was glutathione, carrying a b o u t 70% o f the label present in the organic fraction. I n addition, methionine and cysteine were involved in p h l o e m sulfur transport and accounted for roughly 10%. Primarily, the reduced forms of both, glutathione and cysteine are present in the sieve tubes. Key words: P h l o e m exudate - P h l o e m transport (sulfur) - R i c i n u s - Sulfur - Glutathione.
To make sulfate available for reduction, it is distributed with the transpiration stream and m a y either be reduced at the sites of highest d e m a n d in adequate quantities, or source-sink relationships for reduced sulfur m a y d e m a n d a transport of these c o m p o u n d s . Previously published results (Rennenberg et al. 1979) have shown that tobacco plants export organic sulfur c o m p o u n d s f r o m mature leaves and translocate them t o w a r d sink regions, i.e., the shoot apex, i m m a t u r e leaves, and the r o o t system. This transport occurred at least partially in the phloem. Separation and identification of the organic sulfur translocated revealed that glutathione is the major c o m p o n e n t of the translocate. We report here on transport of sulfate and reduced sulfur c o m p o u n d s in R i c i n u s , with emphasis on the localization o f the translocation p a t h w a y and a m o r e detailed analysis of the translocate f r o m phloem exudate. Material and methods
Introduction
C o r m o p h y t e s cover their sulfur supply nearly exclusively by sulfate uptake f r o m the soil. Since mainly reduced sulfur is b r o u g h t into organic binding, oxidized sulfur has to be reduced before it can be incorporated into c a r b o n skeletons. T h o u g h roots are able to reduce sulfur to some extent (Pate 1965; Ellis 1969), by far m o r e reduction takes place in the leaves, most likely in the chloroplasts (Schwenn and Trebst 1976; Schmidt 1979; A n d e r s o n 1980). * Present address: Max-Planck-Institut ffir Zfichtungsforschung,
D-5000 K61n 30 Federal Republic of Germany ** To whom reprint requests should be sent Abbreviations: CySH=cysteine; GSH=glutathione; GSSG=glutathione disulfide; NEM =N-ethylmaleimide CyS-SCy=cystine
0032-0935/82/0155/0082/$01.40
Plant material and experimental conditions. Ricinus communis L. var. gibsonii Nichols. were grown as pot plants in a heated glass-
house (18-20~ C at night, up to 30~ C at daytime) under natural light conditions, supplemented by additional illumination (Osram L Flnora 65 W, 12 gmol photons m-Zs -1, 16 h photoperiod). Two days before an experiment was started, eight- to eleven-week-old, vigorously growing plants were brought to the laboratory and adapted to the experimental conditions (200-250 gmol photons m-Zs 1, 16 h photoperiod, 25 27~ C). All but the very young leaves at the shoot apex and one mature leaf halfway along the stem were removed. It was observed that the general distribution pattern for leaf fed 3sS was the same in intact plants and in plants having all but one mature leaf detached (experiments not presented). In the present experiments, the plants were prepared in such a way as to reduce the complexity of the transport system. Experiments were started, at the earliest, 48 h after the leaves were cut ; a serious impact of wounding on long-distance transport was not observed. Phloem exudate was collected with graduated glass capillaries from diagonal incisions across a bark strip, which remained intact
U. Bonas et al. : Phloem transport of sulfur
83
after the internode of the fed leaf or the next internode below had been partially girdled at least one day before an experiment was started (c.f. Smith and Milburn 1980). 35SO2 (185 MBq/mgS) was supplied to the leaf using the flap-feeding technic introduced by Biddulph (1941).
Analysis of tissue samples. At the end of an experiment, the stem and petiole were rapidly dissected into 40 m m pieces, frozen and lyophilized. An aliquot of powdered dry weight of each sample was deproteinized (heat denaturation), and 3SS-labeled c o m p o u n d s were extracted with distilled water. Glutathione (GSH) was oxidized to glutathione disulfide (GSSG) according to Jones and Carnegie (1971). The combined water extracts containing GSSG and other 35S-labeled c o m p o u n d s were frozen and lyophilized. The dry residue was resuspended in distilled water and the radioactivity determined by liquid scintillation counting (LSC). Sulfate was precipitated with BaClz at 4 ~ C followed by repeated coprecipitation with N % S O 4. TSe radioactivity of the organic sulfur c o m p o u n d s in the supernatant was measured by LSC. [35S]sulfate activity was calculated. Prior to a chromatographic separation of the organic sulfur compounds, samples had to be further purified by an ion exchange column (Dowex 50 W x 4, 50-100 mesh). 3~S-compounds were washed from the column with 10 N NH3. This effluent was evaporated to dryness, resuspended in 0.2-1 ml distilled water, and further separated using thin-layer chromatography or thinlayer electrophoresis as outlined below. 35S-labeled protein and protein-bound sulfur c o m p o u n d s were determined after sulfate and low molecular weight organic sulfur c o m p o u n d s had been removed: Sulfur c o m p o u n d s b o u n d to protein via disulfide bridges were washed off with borate-buffered dithioerythritol (0.1%, pH 8.6). The residue was treated with 1.5% N a O H to dissolve the protein. Radioactivity of both, protein-bound c o m p o u n d s and protein, were determined by LSC.
Analysis of blade samples. Tissue samples of the fed blade (lamina, veins and petiole), up to 650 mg dry weight, were homogenized at 4 ~ C with 5 ml 80% ethanol and 25 m M N-ethylmaleimide (NEM). During a reaction time of 60 min at 25 ~ C, the substances
with sulfhydryl groups formed stable alkyl derivatives (Gregory 1955). The homogenate was centrifuged for 20rain at 10,000g. The sediment was extracted with 25 m M N E M in 80% ethanol followed by two water extractions, the first at I00 ~ C, and the second at 20 ~ C. The combined extracts were evaporated to l - 2 ml. Further qualitative and quantitative analysis was the same as described for samples of the stem and analysis of exudate, respectively.
Analysis of exudate. Samples of exudate were collected in 10-rain batches for several hours, beginning 20-30 min after [35S]sulfate feeding was started. The volume of exudate was determined and immediately mixed with a three-fold volume of 25 m M N E M in 80% ethanol. After a 60-min incubation at 25 ~ C and subsequent centrifugation, radioactivity of the supernatant containing [3 SS]sul_ fate and 3SS-labeled organic c o m p o u n d s was measured. [3SS]sulfate was separated from other labeled c o m p o u n d s by thin-layer electrophoresis on cellulose (cellulose: M N 300, 0.1 m m thick; verona1 buffer: 5,5-dietbylbarbiturate sodium salt, Serva, 0.075 M, p H 8.6; 600 V; 25 rain). The same system was applied for 90 min to separate the fraction of low molecular weight, 3SS-labeled compounds. Alternatively, these c o m p o u n d s were separated by thin-layer chromatography on cellulose plates ( M N 300), one dimensionaly. Solvents: n-butanol:acetic a c i d : H 2 0 (60:15:25) (by vol.) or two runs in n-propanol: formic acid : H 2 0 (65 : 1 : 34) (by vol.). The radioactivity of separated c o m p o u n d s was recorded with a thin-layer scanner, or the adsorbent containing the substance was scraped from the plate and measured by LSC. Substances were identified by cochromatography and rechromatography with authentic compounds.
Results
Movement o f 35 S in castor bean plants. When [3 s S]sulfate was supplied to a mature leaf by flap-feeding via two major veins for 2 h, 35S was exported toward the shoot apex as well as the root. An example is given in Table 1. Reduction of sulfate, presumably
Table 1. Distribution of 35S in stem and roots of Ricinus communis after a 2 h translocation periode. 35SO 2- was continuously supplied to a mature leaf. The topography of sampling a n d the distribution of 35S-labeled organic c o m p o u n d s and sulfate are shown Dry weight
kBq c m - 1 stem Sulfate
175 198 246 225 333 333 419 397 524 522 642 685 926 706 626 644 6,480
14.38 3.95 1.8i 1.60 2.78 3.70 5.10 3.94 3.97 2.44 t .98 1.81 1.77 2.03 i .88 3.27 56.70
Reduced soluble sulfur 7.04 1.42 1.27 1.00 1.64 2.29 3.19 2.04 2.31 1.63 1.33 1.12 1.00 1.02 0.78 0.63 10.23
Reduced sulfur % of total soluble 35S
kBq.100mg dryweight-1
Sample topography
Sulfate
Reduced solubIe sulfur
Protein
32.9 26.4 41.2 38.5 37.1 38.2 38.5 34.1 36.8 40.0 40.2 38.2 36.1 33.4 29.3 16.1 15.3
8.22 7.98 3.03 2.85 3.34 4.44 4.87 3.97 2.95 1.87 1.30 1.05 0.96 i. 15 1.20 2.44 0.87
4.03 2.87 2.12 1.77 1.97 2.75 3.05 2.05 1.72 1.25 0.88 0.65 0.54 0.58 0.49 0.47 0.16
0.83 0.23 0.11 0.11 0.14 0.18 0.17 0.12 0.10 0.08 0.06 0.05 0.04 0.04 0.03 0,03 0.01
~3 II
84 Q)
Ii
C)
2----@ ii
O
I~ I! ii
O C) Q)
84
U. Bonas et al. : Phloem transport of sulfur
Table 2. Budget of the uptake, reduction, and distribution of 35S in a Ricinus plant during a 2 h period of continuous feeding of 35SO~- to a mature leaf. All other leaves were removed 48 h before the experiment was started. Radioactivity is given in kBq- 102 Sample
Water soluble
Non-water soluble
Total 3sS
% of 35Suptake
Sulfate
Reduced sulfur
Protein
Protein b o u n d sulfur compounds
527.3 1.138
51.02 0,279
6.601 0.026
8,767 0.012
593.688 1.455
98.81 0.24
Export acropetally stem
1.759 0.556
1.065 0,306
0.200 0.022
0.094 0.022
3.118
0.52
shoot apex with youngest leaves
1,203
0.759
0. I78
0.072
Export basipetally stem root
1.737 0.858 0.879
0,720 0,516 0,204
0.047 0.033 0.014
0.100 0,050 0.050
2.604
0.43
600.865
100.00
Fed leaf lamina petiole
Whole plant
585.018
in the chloroplasts, provided appreciable radioactivity confined to low molecular weight substances which moved out of the source leaf. Besides, [35S]sulfate was translocated and distributed acro- and basipetally. There was usually a gradient of decreasing radioactivity from the source leaf to the shoot apex as well as to the root. The percentage of reduced sulfur ranged between 15 and 40% of the total water soluble radioactivity. This relation was relatively constant in the stem (38-40%), but smaller in the shoot apex (33%) and the root system (33-15%). The root and shoot apex contained more [35S]sulfate than the stem pieces. These observations basically remain the same whether they are related to the length of transport pathway or the dry weight (Table 1). It appears, however, that the shoot apex during the 2-h experiment slightly accumulated sulfate and reduced sulfur compounds. The total uptake and distribution of 35S in the plant is shown in Table 2. During the 2-h feeding period, which was the total experimental time, 1.43 ml [35S]sulfate soiution with a radioactivity of 63.9 MBq was absorbed. Approximately 99% of this radioactivity remained in the fed leaf whereas only 1% was exported to the stem. Roughly the same amount of radioactivity was directed toward the shoot apex and the root. Nearly 12% of the absorbed [35S]sulfate was reduced within the 2 h and detected in low molecular weight organic substances, protein, and proteinattached sulfur compounds. Analysis of the water-soluble 35S-compounds of stem segments by thin-layer chromatography revealed that apical to the fed leaf, 77% of the radioactivity in this fraction was confined to glutathione, 5.6% to cysteine, 4% to methionine; below the source leaf
15.847
76% accounted to glutathione, 8.8% to cysteine, and 3.6% to methionine (average of 7 separations).
Distribution of radiosulfur in the leaf. To obtain further information about the loading of organic and inorganic sulfur compounds into the long-distance transport pathway, a leaf was flap-fed with [35S]sulfate via the midvein for 60 min while, at the same time, phloem exudate was collected from the internode below. Thereafter, samples of the lamina, the veins, and the petiole were analyzed for the distribution of [35S]sulfate and reduced radiosulfur. Lamina samples and the veins contained 4-5,4% reduced 35S, whereas the petiole exhibited up to 21% reduced asscompounds. There was an increasing gradient of radioactivity along the petiole, indicating that a lower percentage of reduced sulfur entered the petiole (17 %) and that a higher percentage was loaded into the long-distance transport system of the stem (21%). The composition of the organic sulfur compounds in the leaf is shown in Table 3. Following the path of these compounds from the mesophylI to the veins, along the veins to the petiole, and along the petiole to the transport system of the stem, it may be noted that methionine comprises a much higher radioactivity in samples of the mesophyll and the veins than in the petiole, whereas cysteine shows just the opposite. The reduced form of glutathione also shows a higher label of radioactivity in the petiole than in the mesophyll and the veins, whereas oxidized glutathione carries higher 35S-labeling in the mesophyll and vein samples than in the petiole. The experimental data presented so far indicate that sulfate as well as organic sulfur compounds are phloem mobile. At least export through the petiole
U. Bonas et al. : Phloem transport of sulfur
85
Table 3. Distribution of 35S a m o n g organic sulfur c o m p o u n d s in samples of lamina, veins, and petiole, separated by thin-layer electrophoresis (Met methionine; C y S H = cysteine; G S H = g l u t a t h i o n e ; G S S G = g l u t a t h i o n e disulfide; CyS-SCys=cystine Sample
% of reduced sulfur c o m p o u n d s
Lamina Veins
Met
CySH
13.0 11.5
6.4 11.1
3.6 5.3
12.1 7.0
Petiole upper half lower half
CyS-SCy
GSH
GSSG
Unidentified
5.7 5.0
28.9 31.2
28.1 25.7
17,9 15.5
13.1 15.3
39.0 39.7
20.8 21.5
11,4 I 1.2
and along the stem toward the root point to a transport in the phloem, but translocation up toward the shoot apex could involve phloem as well as xylem transport.
O
~
4 "7
•
/
g-~2
O
/~
0 ~
e
i
40
A__A~A~ e
~
r
60
i
80
9 F
e
e
i
i
e
e
i
e
r
D
100 120 140 160 180 Time (min)
Fig. 1. Releaseof 35S-labeledorganic compounds (A) and [35S]sulfate (o) with phloem exudate. A mature leaf was continuously fed with 35SO4Z-. Collection of phloem exudate started after 35 min at a site 34 cm from the source leaf
Detection of translocated 35S-labeled compounds in phloem exudate. Analysis of translocated compounds shown above is based on extracts of stem segments and blade samples; however, the distribution of 35S obtained in this way must not necessarily be identical to the real composition of the translocate. A better localization of sulfur transport and better characterization of sieve tube content was expected from phloem exudate analysis. An experiment in which a mature leaf of a Ricinus plant was continuously fed with [3SS]sulfate and collection of phloem exudate started 35 rain later is presented in Fig. 1. The collecting site was 34 cm below the source leaf. 35S was already detected in the first
Table 4. Exudate analysis of Ricinus communis after continuous feeding with 35SO~- to mature leaf. Exudate collection began 25 rain after the experiment was started Experiment time (min)
Volume (~d)
25 35 35- 45 45 55 55- 65 65- 75 75- 85 85 95 95-105 105 115 115-125 125 135 135-145 145 155
42 70 85 123 60 90 110 125 120 120 145 161 135 151 i28 115
155-165
165 175 175 185
kBq. 10 gi -I 35SO2-
0.01 0.59 1.16 1.52 1.83 2.23 2.52 2.03 1.75 1.72 2.15 2.08 2.26 2.19 1.71 2.31
Reduced sulfur
Reduced sulfur (% of solubles)
0 0.03 0.18 0.38 0.58 1.19 0.84 0.96 1.06 1.14 0.82 0.98 0.94 0.68 0.78 0.62
0 4.8 13.4 20.0 24.1 34.8 25.0 32.1 37.7 39.9 27.6 32.0 29.4 23.7 31.3 21.2
% of 3sS in reduced sulfur c o m p o u n d s Met
CySH
CyS-SCy
GSH
GSSG
Unidentified
17.0 8.7 7.0 4.2 3.7 3.4 4.0 3.7 3.7 3.6 3.3 2.8 3.5 3.2 2.8
22.0 18.9 11.0 9.8 7.2 6.7 6.9 6.4 4.8 5.6 4.9 3.5 4.0 3.6 3.1
9.0 6.7 3.0 2.0 1.8 1.9 2.3 1.7 2,2 1.8 1.9 1.8 2.9 2.0 2.0
27.6 42.3 45.8 50.1 45.6 48.4 43.5 45.8 46.1 44.2 43.1 42.0 35.6 37.6 34.9
7.7 13.9 22.2 20.8 24.4 24.0 26.3 24.7 25.2 25.2 26.8 29.1 29.0 29.6 31.1
16.7 9.5 11.0 13.1 17.3 15.6 17.0 17.7 18.0 19.6 20.0 20.8 25.0 24.0 26.1
86
U. Bonas et al. : Phloem transport of sulfur ,o-. o
350 300
o
\
250 "7
o
o
200
0 x
O" m
150
o o
100 50
Af
o
30
A/
l
60
9'0
189
150 180 Time(min)
Fig. 2. Pulse feeding experiment. 35SO2- was fed for 3 min followed by a chase periode of 180 min. The profiles of 35S-labelled organic compounds (A) and sulfate (o) released with phloem exudate are shown. The collecting site was 34 cm below the labeled leaf 10-rain batch of exudate, indicating a transport velocity of at least 60 cm h - t . Radioactivity steadily increased with time for 180 min (Table 4). There was a steep increase of [35S]sulfate which finally reached a plateau after 120 min, whereas labeled organic sulfur compounds steadily increased in radioactivity. The rate of exudation varied considerably between 4 and 18.5 gl min 1; within 3 h, almost 1.8 ml exudate were collected containing 1.75% ass absorbed by the leaf. Pulse feeding of [3SS]sulfate for 3 min with a subsequent chase periode of 180 min feeding cold salt solution (Murashige and Skoog 1962), resulted in a release of radioactivity detected 40 min later in the exudate collected 34 cm below (Fig. 2). While reduced, labeled sulfur compounds in the exudate steadily increased with time, the profile for [3SS]sulfate showed a sharp peak, reaching a maximum between 60 and 70 min after the pulse was given. A representative record of 3sS release based on phloem exudate analysis from a plant that was continuously fed is presented in Table 3. Exudate was collected for 185 rain and 10-min batches were analyzed. The exudation rate again varied between 4.2 and 16.1 lal min-1. It was low in the beginning but gradually increased to a level of 13 gl min-1 within the first 2 h of the experiment and subsequently remained constant. [3SS]sulfate as well as reduced sulfur compounds were detected in the phloem sap with both fi'actions exhibiting saturation kinetics. Radioactivity
of the reduced sulfur fraction ranged between 25 and 40% of soluble 3sS. Separation of organic sulfur compounds of phloem exudate by thin-layer electrophoresis revealed the following, clearly identified, radioactively labeled compounds: methionine, cysteine, cystine, reduced, and oxidized glutathione. The highest percentage of 35S was confined to glutathione. The reduced form (GSH) revealed on an average 4 3 % , the oxidized form (GSSG) 26% of asS-labeled soluble organic compounds. In other experiments not presented here, up to 85% of the radioactivity was confined to glutathione. The percentage of 35S in methi0nine was 3.5%, in cysteine 6%, and in cystine 2%. From 60 min on, fluctuation of the percentage distribution among these compounds was minor. A rather high percentage of radioactivity on the thin-layer plates was not identified, 3-4% of the non-identified compound(s) are due to substance(s) which did not chromatograph, but remained at the origin. Discussion
The source of sulfur for the synthesis of many essential sulfur compounds in cormophytic plants is inorganic sulfate taken up through the root. Sulfate is rapidly distributed with the transpiration stream throughout the plant (Biddulph et al. 1958). Pulse feeding experiments with bean plants revealed that 35S applied to the root first accumulated in mature leaves. Twelve - twenty-four hours later, such leaves had lost their radioactivity to the young leaves at the stem apex, indicating an export of 3sS (Biddulph et al. 1958). When 35S was fed to a bean leaf using the flap-feeding technic (Biddulph 1941), radioactivity moved out of the fed leaf and down the stem, mainly through the phloem, as shown by histoautoradiography (Biddulph 1956). Phloem mobility of sulfur was also reported by Weigl and Ziegler (1962), Peel (1970), Rennenberg et al. (1979), Garsed and Mochrie (1980) and is shown here for Ricinus. Movement down the stem usually excludes movement in the xylem, whereas upward movement might either be localized in the xylem, in the phloem, or in both since a lateral exchange cannot be excluded. One might argue that sulfur, introduced by a leaf flap and assimilated in the mesophyll, is passively swept out of the leaf with the assimilate stream whenever sulfur compounds enter the sieve tubes. The analysis of 3sS in the lamina and along the conducting pathway revealed, however, that the relative amount of reduced organic sulfur compounds in the lamina was much less (9%) than in the petiole (20%) and in the stem (36%), which indicates that preferably organic sulfur compounds may be loaded into the sieve elements (c.f. Table 2). Though a high accumula-
U. Bonas et al. : Phloem transport of sulfur
tion capacity of phloem, especially of sieve elements, for ions (phosphate, sulfate) has previously been reported (Bieleski 1966), the present experiments indicate a selective permeability, favoring the entrance of reduced sulfur compounds over sulfate. It is assumed that the distribution of ass follows source-sink relationships for reduced sulfur compounds and sulfate. The directionality is probably not determined solely by concentration gradients for sulfur compounds, but by an overall concentration gradient for ions, carbon, nitrogen-, and sulfur assimilates. Transport of ass in the phloem was clearly demonstrated here by phloem sap analysis. Release of phloem exudate from bark incisions of Ricinus was reported before, and circumstantial evidence was presented that phloem exudate represents sieve tube content (Milburn 1971 ; Hall et al. 1971 ; Hall and Baker 1972; Smith and Milburn 1980). Under this assumption the chemical composition of the sap should reflect the translocate. The analysis of phloem exudate of Ricinus revealed that the main radioactivity was confined to inorganic sulfate. Sulfate is known to be an anionic component of Ricinus phloem exudate though its concentration is rather low (0,024-0,048 mg m l - 1) (Hall and Baker 1972). Sulfate is considered to play a minor role in phloem transport of sulfur as compared to the sulfur movement in the organic form (Ziegler 1975). This is in contradiction to results presented here, demonstrating that only 30-40% of ass in the translocate is in organic binding. The conclusive answer to this question can only be obtained by a determination of the specific radioactivity; the absolute quantities of sulfate and sulfur amino acids, however, were not measured. It is only known that methionine is present in trace amounts in sieve tube sap (Hall and Baker 1972). The percentage distribution of radiosulfur among sulfate and organic sulfur compounds was nearly the same in stem segments and phloem exudate, indicating that the ass-labeled translocate probably did not leave the long distance transport channel or was not further metabolized after it might have left the sieve tubes (c.f. Tables 2, 4). The main labeled organic compound in phloem sap of Ricinus is glutathione. The reduced form (GSH) revealed a much higher radioactive labeling than the oxidized form (GSSG) (Table 4). Provided that the specific activity of glutathione is more or less constant after 60 min, the absolute amount of GSSG is not 50% that of GSH, as indicated by Table 4, but only one quart, since one molecule GSSG carries two sulfur atoms. It seems that glutathione is translocated in the reduced form. This was further confirmed by electrophoresis of exu-
87
date immediately treated with N E M to prevent oxidation. Therefore, the oxidized form of glutathione (GSSG) ranged between 25 and 35% total glutathione-sulfur, the percentage of cystine (CyS-SCy) was 30-40% total cysteine-sulfur, indicating that the reduced substances are the favored transport forms. The present experiments support the idea that green cells of higher plants are able to reduce more sulfate than necessary for their own needs and to incorporate this surplus into glutathione that is translocated to places of high demand, i.e., the apex and the root system (Rennenberg et al. 1979). Though the experiments were not designed to measure exact translocation velocities, rough figures can be given even though translocation velocity is variable and dependent on a number of exogenous and endogenous factors. Velocity of asS-export of leaf-fed sulfate was 30 cm h -1 in " i n t a c t " plants (Table 1) and at least 58 cm h -1 in exuding stems (Fig. 1, 2). Hall et al. (1971) reported a velocity of > 100 cm h 1 for the translocation of ~4C-labeled assimilates in exuding Ricinus plants and 80-84 cm h - ~ in intact plants. Translocation velocities > 63 cm h - 1 were derived by Smith and Milburn (1980) from measurements of mass transfer. It appears that transport velocity is higher in exuding plants as compared to intact plant systems. This apparently has an impact on translocation rate. Although we have not determined the specific mass transfer, our results indicate that sulfur transport might be more efficient in the exuding stem than in intact plants. The results presented in Table 1 and Table 2 show that in an intact Ricinus plant roughly 1% of total ass absorbed moved out of the fed leaf and along the stem toward the root and the apex. In the exuding plant (Fig. 1), 1.7% were collected with the phloem sap, indicating a higher export if movement of ass toward the shoot apex is taken into account. These results may be explained by the assumption that bark incisions create a high, artificial sink, attracting most of the translocate. The specific mass transfer of the enhanced phloem transport in exuding plants must have been quite high, since the conducting cross-sectional area of phloem was reduced to approximately 30% by partial girdling of the stem. Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.
References Anderson, J.W. (1980) Assimilation of inorganic sulfate into cysteine. In: The biochemistry of plants, vol. 5, pp. 203 223, Miflin, B.J., ed. Academic Press, New York Biddulph, O. (i941) Diurnal migration of injected radiophosphorus from bean leaves. Am. J. Bot. 28, 348 352
88 Biddulph, S.F. (1956) Visual indications of S 35 and p32 translocation in the phloem. Am. J. Bot. 43, 143-148 Biddulph, O., Biddulph, S.F., Cory, R., Koontz, H. (1958) Circulation patterns for p~2 S3S and Ca 45 in the bean plant. Plant Physiol. 33, 293-300 Bieleski, R.L. (1966) Sites of accumulation in excised phloem and vascular tissues. Plant Physiol. 41, 455 466 Ellis, R.J. (1969) Sulfate activation in higher plants. Planta 88, 3442 Garsed, S.G., Mochrie, A. (1980) Translocation of sulphite in Vicia f a b a L. New Phytol. 84, 421 428 Gregory (1955) The stability of N-ethylmaleimide and its reaction with sulfhydryl groups. J. Am. Chem. Soc. 77, 3922-3923 Hall, S.M., Baker, D.A. (1972) The chemical composition of Ricinus phloem exudate. Planta 106, 131 140 Hall, S.M., Baker, D.A., Milburn, J.A. (1971) Phloem transport of 14C-labelled assimilates in Ricinus. Planta 100, 200-207 Jones, J.K., Carnegie, P.R. (1971) Binding of oxidized glutathione to dough proteins and a new explanation, involving thioldisulfide exchange, of the physical properties of dough. J. Sci. Food. Agric. 22, 358-364 Milburn, J.A. (1971) An analysis of the response in phloem exudation on application of massage to Ricinus. Planta 100, 143-154 Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. lfi, 473 497
U. Bonas et al. : Phloem transport of sulfur Pate, J.S. (1965) Roots as organs of assimilation of sulfate. Science 149, 547 548 Peel, A.J. (1970) Further evidence for the relative immobility of water in sieve tubes of willow. Physiol. Plant. 23, 667 672 Rennenberg, K., Schmitz, K., Bergmann, L. (1979) Long-distance transport of sulfur in Nicotiana tabacum. Planta 147, 57-62 Schmidt, A. (1979) Photosynthetic assimilation of sulfur compounds. In: Encyclopedia of plant physiology, N.S., vol. 6, pp. 481496, Gibbs, M., Latzko, E., eds. Springer, Berlin Heidelberg New York Schwenn, J.D., Trebst, A. (1976) Photosynthetic sulfate reduction by chloroplasts. In : The intact chloroplast, pp. 315 334, Barber, J., ed. Elsevier, Amsterdam New York Oxford Smith, J.A.C., Milburn, J.A. (1980) Phloem transport, solute flux and the kinetics of sap exudation in Ricinus communis L. Planta 148, 35-41 Weigl, J., Ziegler, H. (1962) Die rfiumliche Verteilung von 3sS und die Art der markierten Verbindungen in Spinatblfittern nach Begasung mit 35SO2. Planta 58, 435 447 Ziegler, H. (1975) Nature of substances in the phloem. In: Encyclopedia of plant physiology, N.S., vol. 1, pp. 59-100, Zimmermann, M.H., Milburn, J.A., eds. Springer, Berlin Heidelberg New York
Received 6 February; accepted 4 March 1982
