Sink to Source Translocation in Soybean - Plant Physiology

Plant Physiol. (1984) 74, 434-436 0032-0889/84/74/0434/03/$O1.00/0

Short Communication

Sink to Source Translocation in Soybean' Received for publication September 22, 1983

ALAN B. BENNETT*2, BARRY L. SWEGER3, AND ROGER M. SPANSWICK Section of Plant Biology, Division of Biological Sciences, Cornell University, Ithaca, New York 14853 ABSTRACE The possibility that phloem loading may occur in the reproductive sink tissues of soybeans (Glycine max Meff. cv Chippewa 64) was examined. When I14Clsucrose was applied to seed coat tissues from which the developing embryo had been surgically removed, 0.1% to 0.5% of the radioactivity was translocated to the vegetative plant parts. This sink to source translocation was largely unaffected by destroying a band of phloem with steam treatment on the stem above and below the labeled pod. The same steam treatment, however, completely abolished translocation of I14Cisucrose between mature leaves and developing fruits. These results indicate that the movement of nutrients from developing seed coats to the vegetative plant parts occur in the xylem and that phloem loading does not occur in this sink tissue.

In recent years, several studies have provided evidence suggesting that processes localized in sink tissues are important in regulating the partitioning of assimilates to storage tissues (4, 7, 13, 15). This realization has led to detailed examinations of the mechanisms of nutrient uptake by sink tissues (2, 7, 8, 10, 12) and to the development of techniques to monitor phloem unloading in the sink tissue of developing legume fruits (11, 14). One model of phloem unloading is that unloading occurs by passive solute leakage from the phloem wherever active uptake by the phloem is impaired. This model further proposes that sinks locally inhibit the phloem loading mechanism and thereby affect net unloading of the phloem (5). However, evidence has recently been presented indicating that phloem unloading does not occur by a passive leakage of solutes but that it occurs by an energy-dependent and possibly carrier-mediated process (11, 14). In order to test these two models of phloem unloading, we have examined directly whether phloem loading could occur in soybean seedcoat tissue after removal of the developing embryo. These experiments utilized the techniques developed by Thorne and Rainbird (11) whereby the metabolic sink tissues (developing embryo) can be surgically and nondisruptively removed from the site of phloem unloading (seed coat). This allowed a direct examination of the potential for phloem loading to occur in the seed coat when no longer under the influence of the sink storage tissue.

MATERIALS AND METHODS Soybeans (Glycine max Merr. cv Chippewa 64) were grown in a greenhouse with supplemental lighting. Plants were watered daily and fertilized weekly with liquid fertilizer (Peter's 20-2020). Plants were brought to the laboratory from the greenhouse when fruits at nodes nine through twelve contained partially developed seeds. All experiments were carried out in the laboratory. Access was gained to the seed coat tissue of a central seed in three-seeded pods by surgically opening the pod and removing the developing embryo as described by Thorne and Rainbird (11). This procedure left half of the seed coat intact and still attached to the pod wall. The seed coat half was filled with a salt solution (approximately 25 ,l) containing 0.5 mm KCl, 0.5 mM CaCl2, 0.1 mM MgCl2, and 5 mm Mes adjusted to pH 6.0 with NaOH, and the pod subsequently was wrapped with parafilm to prevent dehydration. After 1 h, the salt solution was replaced and supplemented with 4.2 gCi ['4C]sucrose (New England Nuclear, 3.7 Ci/mol). The salt solution was replenished every hour and, after 4 h, the plant was severed at the base and the plant parts lyophilized, oxidized in a Packard Tri-Carb B306 sample oxidizer, and the radioactivity determined by liquid scintillation spectroscopy. Counting efficiency was approximately 42%. Steam girdling of the stem was performed 12 h prior to the start of labeling and was accomplished with a fine jet of steam directed at a 0.5-cm band of the main stem for 1 min. A splint was fashioned to ensure that the structural integrity of the steamgirdled stem was not impaired. Labeling of a mature leaf with ['4C]sucrose was accomplished by first abrading the upper leaf surface with a carborundum/ water paste. The ['4C]sucrose (4.2 MCi) was applied to the abraded surface within a ring of silicon vacuum grease that was subsequently covered with a glass coverslip. Four h after labeling of the leaf, a single fruit at an intermediate stage of development was collected from each node and assayed for radioactivity as described above. RESULTS Experiments in which a developing seed coat at node 9 was labeled with ['4C]sucrose (experiment as diagrammed in Fig. lA) and the entire plant subsequently monitored for radioactivity indicated that 0.1 % to 0.5% of the applied 14C was translocated to the vegetative plant parts (Table I). Approximately 80% of the translocated radioactivity was recovered in the leaves with lesser amounts recovered in the internodes, petioles, and developing fruits (Table I). Of the radioactivity translocated to the leaves, the largest amounts were found at the 2nd and 4th node above the labeled pod (data not shown). Since soybean leaves at every other node share a common vascular system (1) this pattern of translocation from sink to source suggested that solute movement was occurring through the vascular system.

Supported by the United States Department of Agriculture Competitive Grants Program grant No. 81-CRCR-1-0758. 2 Present address: Department of Vegetable Crops, University of California, Davis, California 95616. 3 Present address: Department of Biology, Lebanon Valley College, Annville, Pennsylvania 17003. 434

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SINK TO SOURCE TRANSLOCATION

A

B

C

Table III. Distribution of '4C in Developing Soybean Fruits after 4 Hours ofLabeling of a Mature Leaf at Node 1) with 4.2 MCi of['4C Sucrose Plants used were either untreated (control) or were steam girdled on the main stem above and below node 9. The experiment was performed as described in "Materials and Methods," and the values shown are the mean of two experiments. Fruit at Node Number

Distribution of Radioactivity Control Steam girdled

cpm FIG. 1. Illustration of a soybean plant and the experimental treatments used in this study. Shaded plant organs indicate where ['4C]sucrose was applied, and dashed lines (-- -) indicate where bands of phloem were destroyed by steam girdling. A, The seed coat of a central seed in a developing fruit at node 9 was labeled with ['4C]sucrose, and the aboveground plant parts were subsequently monitored for radioactivity. B, The main stem above and below node 10 was steam girdled --- -), the seed coat of a central seed in a developing fruit at node 10 was labeled with ['4C]sucrose, and the vegetative leaves were subsequently monitored for radioactivity. C, The main stem above and below node 9 was steam girdled (- - -), leaf 11 was labeled with ['4C]sucrose, and the developing fruits were subsequently monitored for radioactivity.

7 8 9 10 11 12 13 14 15

24,350 296 19,591 135 79,748 341 90 44 31

34 31 44 26 39,772 222 51 13 11

Total

124,626

40,204

In preliminary experiments, sink to source translocation was not inhibited by the addition of 1 mM NaCN to the seed coat

labeling solution, a treatment expected to inhibit phloem loading. Additional experiments indicated that treatments which reduced transpiration (darkness and low temperature) also reduced sink to source translocation (data not shown). Together these results suggested that movement of radioactivity from the seed coat may have occurred by mass flow in the xylem. To test this possibility, a band of phloem on the main stem above and below node 10 was destroyed by treatment with steam (steam girdle). An opened seed coat at node 10 was then labeled with ['4C] sucrose, and translocation to the vegetative leaves was determined (experiment as diagrammed in Fig. 1B). The extent and patterns of sink to source translocation were similar in control and steam-girdled plants (Table II), indicating that this movement of radioactivity did not require functionally intact phloem. To ensure that the steam treatment of the stem was effective in disrupting the phloem, the effects of this treatment on transof ['4C]sucrose from a mature leaf to developing fruits Table II. Distribution of '4C in Leaves after 4 Hours ofLabeling of an location determined. In these experiments, the stem above and below Opened Seed Coat in a Developing Soybean Fruit at Node 10 with 4.2 were node 9 was steam girdled, and ['4C]sucrose was applied to the MCi of['4C]Sucrose abraded surface of leaf eleven. After four h, translocation of 14C Plants used were either untreated (control) or steam girdled on the to the developing fruits was monitored (experiment as diamain stem above and below node 10. The experiments were performed grammed in Fig. 1C). Here it was evident that no 14C was as described in "Materials and Methods," and the values shown are the translocated beyond the steam girdle (Table III). Since translomean of two (control) or three (steam girdled) experiments. cation from source to sink is generally regarded as a phloemmediated process, this result demonstrated the effectiveness of Distribution of Radioactivity Leaf at the steam girdle in disrupting the phloem. Node Number Control Steam girdled cpm DISCUSSION 6 320 505 The results of the experiments in which the phloem was 7 176 431 disrupted by steam treatment clearly demonstrated that translo140 202 8 cation from the sink did not require intact phloem and suggested 9 196 181 that sink to source translocation occurred predominantly, if not 462 178 10 comletely, by mass flow in the xylem. This result is consistent 11 268 155 with the findings of Nooden and Murray (9) which indicated 12 336 400 that soybean fruits exerted an influence on leaf senescence via 316 13 261 the xylem. Since mass flow in the xylem is nonselective, this 14 505 163 finding suggests that any seed-derived compounds that are se15 381 132 to the seed apoplast may be translocated to the vegetative creted 16 235 161 parts of the soybean plant where they may exert an influence on vegetative growth and development. These results also demonTotal 3335 2739 strate that mass flow of water and solutes from developing seeds Table I. Distribution of '4C in the Vegetative Plant Parts after 4 Hours of Labeling of an Opened Seed Coat in a Developing Soybean Fruit at Node 10 with 4.2 UCi of['4CJSucrose The experiment was performed as described in "Materials and Methods," and the values shown are the mean of two experiments. Distribution of Radioactivity Vegetative Organ % of total cpm 5166 80 Leaves 5 Petioles 293 11 737 Intemodes 4 Fruits 248 6444 100 Total

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can occur in the xylem and may provide the means by which excess water delivered to the developing seed in the phloem may be recirculated to the vegetative plant parts. Such a recirculation of water has been proposed as a mechanism to remove excess phloem-derived water from developing wheat grains (6). Quantitatively, source to sink translocation from a leaf to a fruit two nodes below was 50- to 100-fold greater than translocation in the reverse direction (compare Table II and III) when measured over a 4-h period. As one would expect, then, this reverse flow of radioactivity from sink to source is nutritionally insignificant. However, as discussed above and demonstrated by Nooden and Murray (9), sink to source translocation may be physiologically important to sink/source interactions. With respect to the mechanism of phloem unloading in soybean seed coats, the results presented here indicate that phloem loading does not occur in this tissue even after removal of the sink storage tissue. This does not support the model of phloem unloading which proposes that solutes leave the phloem by leakage and that sinks locally inhibit the reloading process. By this model, the activity of the reloading process should have been apparent after removal of the metabolic sink (i.e., the developing soybean embryo). Instead, the results presented here suggest that the phloem in the seed coat does not have the enzymic capacity for phloem loading and provides circumstantial support for an alternate model of phloem unloading which involves an active unloading mechanism. Acknowledgments-We thank Dr. Francis Hsu for useful suggestions and stimulating discussions.

Plant Physiol. Vol. 74, 1984 LITERATURE CITED

1. BELL WH 1934 Ontogeny of the primary axis of soja max. Bot Gaz 95: 622635 2. BENNETT AB, RM SPANSWICK 1983 Derepression of amino acid-H+ cotrans port in developing soybean embryos. Plant Physiol 72: 781-786 3. CAMERON-MILLS V, CM DUFFUS 1979 Sucrose transport in isolated immature barley embryos. Ann Bot 43: 559-569 4. EGLI DB, J FRASER, JE LEGGErr, CG PONELEIT 1981 Control of seed growth in soya bean (Glycine max L. Merrill). Ann Bot 48: 171-176 5. GIFFORD RM, LT EVANS 1981 Photosynthesis, carbon partitioning, and yield. Annu Rev Plant Physiol 32: 485-509 6. JENNER CF 1982 Movement of water and mass transfer into developing grains of wheat. Aust J Plant Physiol 9: 69-82 7. JENNER CF 1974 Factors in the grain regulating the accumulation of starch. R Soc N Z Bull 12: 901-908 8. LICHTNER FT, RM SPANSWICK 1981 Sucrose uptake by developing soybean cotyledons. Plant Physiol 68: 693-698 9. NOODEN LD, BJ MURRAY 1982 Transmission of the monocarpic senescence signal via the xylem in soybean. Plant Physiol 69: 754-756 10. SAFrNER RA, RE WYSE 1980 Alkali cation/sucrose co-transport in the root sink of sugar beet. Plant Physiol 66: 884-889 1 1. THORNE JH, RM RAINBIRD 1983 An in vivo technique for the study of phloem unloading in seed coats of developing soybean seeds. Plant Physiol 72: 268271 12. THORNE JH 1982 Characterization of the active sucrose transport system of immature soybean embryos. Plant Physiol 70: 953-958 13. THORNE JH, HR KOLLER 1974 Influence of assimilate demand on photosynthesis, diffusion resistance, translocation, and carbohydrate levels in soybean leaves. Plant Physiol 54: 201-207 14. WOLSWINKEL P, A AMMERLAAN 1983 Phloem unloading in developing seeds of Vicia faba L. The effects of several inhibitors on the release of sucrose and amino acids by the seed coat. Planta 158: 205-215 15. WYSE RE, RA SAFTNER 1982 Reduction in sink-mobilizing ability following periods of high carbon flux. Plant Physiol 69: 226-228