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a Dep. of Agronomy and Plant Genetics, 411 Borlaug Hall, Univ. of Minnesota, 1991 Upper Buford Circle, St. Paul, MN 55108
b Dep. of Agronomy, 1563 Agronomy Hall, Iowa State Univ., Ames, IA 50011
* Corresponding author (naeve002{at}umn.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: DAI, days after imbibition • ICP–OES, inductively coupled plasma–optical emission spectroscopy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Leaf proteins are a major source of remobilizable S and N; however, S cycling is primarily regulated via the protein N cycle (Dangl et al., 2000). Seed protein production puts a large demand on the soybean's vegetative tissues for mobilization of stored nitrogen (N) (Sinclair and DeWitt, 1975; Shibles and Sundberg, 1998) and S (Anderson and Fitzgerald, 2001). Soybean seed-N is composed of approximately one-half mobilized N under normal field growth conditions (Hanway and Weber, 1971; Loberg et al., 1984; Imsande and Edwards, 1988), whereas soybean mobilizes 66 to 79% of its vegetative-N (Vasilas et al., 1995). Proteins found in vegetative tissues are also important sources of remobilizable S (Buchanan et al., 2000). However, little published work has focused on S mobilization in soybean grown under S-sufficient conditions.
Soybean leaf-S concentration declines during seed filling (Sweeney and Granade, 1993; Fantanive et al., 1996; Sexton et al., 1998b), as does the N/S ratio (Sweeney and Granade, 1993). The N/S ratio in leaves drops from 17.2 to 11.5, indicating that approximately 50% more S relative to N remains in abscised leaves than in healthy ones. Soybean vegetative tissues may accumulate ample amounts of S for the production of high quality and high protein seed, but not efficiently mobilize it from vegetative to reproductive tissues (Sunarpi and Anderson, 1997b; Sexton et al., 1998b).
Sulfur is first delivered to the aerial portion of plants as SO4 via xylem (Adiputra and Anderson, 1995; Bell et al., 1995; Sunarpi and Anderson, 1996b). Smith and Lang (1988) reported that S is transported to mature leaves and is quickly exported, unchanged, via phloem. These mature leaves appear to act as transfer stations for S. Mature barley (Hordeum vulgare L.) leaves import SO4, but this S remains in a pool isolated from endogenous S (Adiputra and Anderson, 1995). The latter was probably primarily in the form of protein or sulfolipid. Smith and Lang (1988) reported that 90% of the S transported via the phloem is inorganic.
While mature leaves tend to sequester little of the newly acquired S (Smith and Lang, 1988), it has been shown that expanding leaves are strong sinks for newly acquired SO4 (Smith and Lang, 1988; Bell et al., 1995; Sunarpi and Anderson, 1997a; Anderson and Fitzgerald, 2001). In one study with soybean, 20% of leaf SO4 was reduced to organic forms of S; the remainder was stored as SO4 in the vacuole (Bell et al., 1994). Sunarpi and Anderson (1998) eloquently described S redistribution in S-deficient vegetative soybean. They found that the largest newly expanded leaf plays an important role as an intermediary in the transport of S from the root to the youngest expanding leaves. Additionally, they found that approximately 25% of this mobilized S was recycled via the root as SO4.
Although mobilization of S from vegetative tissues is likely to be important for seed protein synthesis in soybean, very little is known about the timing, quantity, and efficiency of S mobilization to the seed. Sunarpi and Anderson (1997b) examined S mobilization in reproductive soybean. Maximum length of leaves formed de novo during their study period seemed to be inhibited by S deficiency. The authors acknowledge that S was supplied at suboptimal levels (8.3 µmol S supplied plant–1 d–1). Soybean plants supplied with less than 2 mmol S plant–1 d–1 show reduced seed yield, and rate of dry matter and N accumulation in a 130-d growing season (Sexton et al., 1998b).
Sunarpi and Anderson (1997b) reported that soybean leaves did not act as large reservoirs for S. Although total leaf- and pod-S content dropped by 49 and 67% during seed fill, little of the seeds' S needs may have been met from mobilized-S. The plants acquired 87% of the seeds' needs during seed fill. Nearly all of the mobilized leaf- and pod-S came from a discrete soluble-S fraction (primarily SO4). However, the pods' contribution to the total seed-S was small. One might expect plants to metabolize, store, and mobilize S very differently under S sufficiency.
Transgenic approaches to increase methionine and cysteine concentrations within soybean seed have not been particularly fruitful (Sexton et al., 2002). Sulfur-amino acid accumulation in the seed may be limited by delivery of S to the seed. The aim of this research was to determine the uptake, distribution, and mobilization patterns of S during the reproductive phase of soybean development. The effects of timing of S uptake and form of S stored in tissues on S mobilization to developing seed were explored.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Two similar experiments were performed. Differences in plant culture, pulse-chases, and harvests are as follows:
Experiment #1
Seeds were allowed to imbibe water on germination papers on 22 Aug. 1996. Seedlings were transferred to hydroponic medium six d after imbibition (DAI). Seedlings were transferred to single plant containers at 24 DAI.
Nutritive pulses of 35SO4 were administered to one group of plants at 53 DAI (Pulse 1) and a second group at 82 DAI (Pulse 2). These dates approximately corresponded with soybean developmental stages R4.0 and R6.0, respectively. These reproductive stages represent periods before and early in seed growth, respectively. Separate pulses were provided at these stages to examine the relative changes in the uptake, distribution, and mobilization patterns of S through reproductive development.
Plants to be labeled were removed from nutrient solutions three d before pulse feeding. Their roots were rinsed with distilled water, and they were placed in nutrient solutions with 50 µM SO4. Pulse 1 plants received 1980 kBq 35S as Na235SO4, and Pulse 2 plants received 2430 kBq 35S. Because of the high specific activity of the isotope (approximately 40 TBq [mmol SO4]–1), the pulse contributed an insignificant amount of SO4. Plants were pulsed for 3 d. Subsequently, their roots were triple-rinsed with distilled water, four plants were harvested to determine uptake, and the others were returned to fresh 50 µM SO4 nutrient solution. The next Day 0.4 mM SO4 was resupplied.
Plants were harvested at stages R4.0, R5.0, R5.5, R6.0, R7.0, and R8.0. Roots of all plants to be harvested were triple-rinsed. Plants were immediately dissected into roots, stems (including petioles), leaves, pods and flowers, and seeds (when present). Separated plant parts were placed in a forced-air drying oven at 65°C for three d, after which they were weighed and ground. The final harvest was taken at physiological maturity (R8.0). Abscised leaves were collected and pooled with any leaves remaining on the plant.
Quantification of 35S in tissue was by use of a Beckman 3801 liquid scintillation counter (Beckman Coulter, Inc., Fullerton, CA). Tissue discoloration and solubilization was accomplished using sodium hypoclorite in a method adapted from Packard Instrument Company (1996). Briefly, 20 mg powdered tissue was placed in a scintillation vial. One mL of a 5.25% sodium hypoclorite solution was added, and the vials were tightly capped and swirled to wet the tissue. Vials were placed in a forced-air oven at 65°C for 2 h. The vials were cooled to room temperature, vented, and chlorine vapor was removed with a gentle stream of air. Eleven mL of scintillation cocktail solution (Ultima Gold XR, Packard Instrument Co., Meriden, CT) was added to each vial, and the samples were allowed to rest in the dark overnight. They were then counted and corrected for quench and 35S decay.
Quantification of labeled SO4 was by a method adapted from Bell et al. (1994). Briefly, labeled SO4 concentration was calculated by the difference in activity of the total soluble fraction and soluble fraction after a BaCl precipitation of SO4.
Total S was determined by inductively coupled plasma–optical emission spectrometry (ICP–OES). Dried plant material was digested with HNO3 and H2O2 in a microwave digestion system (Model MDS 2000, CEM Corp., Matthews, NC) by a method adapted from Bañuelos and Akohoue (1994) and Sah and Miller (1992). Total S in solution was determined by ICP–OES (Model IRIS/AP DUO, Thermo Jarrel, Franklin, MA,). Nitrogen was determined by micro-Kjeldahl analysis according to the methods of Bremner and Breitenbeck (1983).
Experiment #2
This experiment was conducted in a manner very similar to Exp. 1. Seeds were allowed to imbibe water on germination papers on 12 Mar. 1997. Seedlings were transferred to hydroponic medium at 7 DAI, and then were transferred to single-plant containers at 23 DAI.
Five pulses were administered at 65, 81, 98, 107, and 119 DAI (stage R2.0, R4.0, R5.5, R6.5, and R7.0, respectively) to discrete sets of plants. Plants to be pulsed were removed from the complete nutrient solution one d before the labeling and placed directly in complete nutrient solution without SO4. The following day, H235SO4 was added, with each plant receiving 3017 kBq 35S. Plants were pulsed for ten h. Four plants were immediately harvested after the pulse. Remaining labeled plants were immediately transferred to full nutrient solutions containing unlabeled S. Plants were harvested as in Exp. 1, except that harvests occurred at stages R2.0, R4.0, R5.5, R6.5, R7.0, and R8.0. Additionally, the three most recently opened leaves and any leaf buds were harvested separately from the older, fully expanded leaves. Tissue analysis for Exp. 2 was conducted in exactly the same manner as in Exp. 1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Carpel tissues (hereafter termed simply "pods") were the only fraction that consistently lost S on a concentration basis throughout the study. In Exp. 1, pod tissue averaged 1.9 ± 0.1 mg S (g dry matter)–1 across the first two harvests, and 0.5 ± 0.0 mg S (g dry matter)–1 at maturity. Pods, although pericarp tissue (Esau, 1977), act like vegetative tissues by mobilizing reduced C and mineral nutrients to seed (Thorne, 1979). Although pods did not contain large quantities of S (Fig. 1B, 2B), they mobilized it at approximately 50% efficiency. Leaves did not mobilize S on a concentration basis, or as efficiently as pods; however, they contained greater than 50% of the plant's S before seed fill, and mobilized significant quantities of total S. This was most clearly shown in Exp. 1. Leaves mobilized S and supplied up to 25 and 17%, respectively, of the seed's total S in Exp. 1 and 2. Pods contributed 10 and 8.8%. All vegetative tissues mobilized S to seed and, in all, they supplied 40 and 36% of the respective total seed-S requirement in Exp. 1 and 2.
Initial Distribution of 35S Acquired during Reproductive Development
Pulse 1
In Exp. 1, nearly 60% of the total 35S supplied at R4.0 was found distributed to the leaves (Fig. 3A)
. Roots and stems acquired 20 and 13% of the label, respectively. Pods also contributed to S storage. They contained 8.0% of the label initially, although their dry weight made up only 4.4% of the plant's total dry weight (Fig. 1A).
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Tissues contained varying amounts of 35SO4 immediately after the first pulse in Exp. 1 (Fig. 3C). Labeled SO4 made up only a small portion of the leaf 35S (3.2%) (see Fig. 3A, 3C). Roots and stems contained 7.4 and 15% of their label as 35SO4, respectively, while the pod label was 39% 35SO4. Tissues in Exp. 2 contained larger quantities of unreduced 35S than those in Exp. 1 (Fig. 3C, 4C). Roots and stems were highly enriched in 35SO4 (42 and 53% of total tissue label, respectively). Leaves also retained a much larger portion of their total label as 35SO4; however, 35SO4 still amounted to less than 20% of the total label. Differences between experiments in initial 35SO4 likely were attributed to the shorter labeling time used in Exp. 2. In addition, plants in Exp. 2 had a lower demand for SO4, and tended to utilize SO4 less rapidly and retain a larger fraction of their radiosulfur as 35SO4 throughout the study. Due primarily to higher densities and increasing day lengths, plants in Exp. 2 grew slower and attained a smaller mature dry weight (Fig. 2A) and total S content (Fig. 2B).
Pulse 2
A second pulse was administered to a different set of plants at R6.0 and R5.5 (DAI 82 and 98) in Exp. 1 and 2, respectively. This corresponded to the beginning of rapid seed filling (Fig. 1A, 2A). In Exp. 1, seeds were heavily labeled after the pulse period (25% of the total plant label, while making up just 7.8% of the total dry matter), whereas the leaf fraction contained just 14% of total plant 35S (Fig. 3B). Pods contained 18%, while roots and stems incorporated 29 and 15% of whole-plant label, respectively. Roots and stems contained approximately 10% and pods contained approximately 40% of their total label as 35SO4 immediately after the pulse (Fig. 3B, 3D). These values were much higher (50 to 67%) in Exp. 2 (Fig. 4B, 4D).
Although the seed portion of the plant total dry weight after Pulse 2 of Exp. 2 was nearly identical to that in Exp. 1 (Fig. 1A, 2A), this seed fraction contained almost no 35S immediately after the pulse in Exp. 2 (Fig. 4B). Alternatively, the leaf and root fractions were more heavily labeled than in Exp. 1. Again, these differences appear to have been due to the shorter pulsing period in Exp. 2.
Additional Pulses
Three additional pulses were administered as separate treatments in Exp. 2. Five distinct pulses were given to better identify uptake and mobilization patterns for S acquired by soybean throughout reproduction. Developmental changes during this time period had a large effect on the initial sink strength of individual tissues for newly acquired S.
Leaves acquired a slightly smaller portion of the total label from the pulse at each subsequent labeling (initial leaf fraction, Fig. 5) . Although leaf dry weight continued to increase through 119 DAI, leaf dry weight accumulation rate slowed after 98 DAI. This corresponded with a large decrease in S uptake by leaves. Pods and seeds each acquired a larger portion of the initial plant label during subsequent pulses (initial pod fraction and initial seed fraction, Fig. 5). Stems acquired 15 ± 1% of the label at each date, while roots retained the balance (data not shown). Initial S distribution appears to reflect the changing growth rates of these tissues as the plants passed from vegetative to reproductive development. Seeds, however, received no more than 10% of the total plant label during any of the 10 h pulses given through late seed development.
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All vegetative tissues acquired 35SO4 that was later mobilized as SO4 or reduced to labeled organic-S before mobilization with approximately 90% efficiency (Fig. 3C, 4C), with the exception of the root fraction in Exp. 1 (Fig. 3C). There, the roots retained a small amount of 35SO4 that was not replaced by unlabeled-S during the chase period where only unlabeled S was supplied to the nutrient solution. In Exp. 2, the developing pods took up SO4 indirectly after the pulse and reduced or mobilized 85% of this before maturity (Fig. 4C). Labeled organic-S was mobilized in all tissues less efficiently than 35SO4 (Fig. 3C, 3E, 4C, 4E).
For whole plants, very little 35SO4 was lost or reduced after the first harvest in Exp. 1 (equal to 2.0% of the initial plant label), whereas 23% of the initial plant label was reduced after the pulsing period but before maturity in Exp. 2. As with Exp. 1, much of this reduction occurred by the second harvest, with the remainder occurring principally by early seed filling.
Seed tissue acquired labeled-S from the beginning of seed development, paralleling uptake of total-S (Fig. 1B, 2B). This acquisition continued as vegetative tissues mobilized their label through maturity. In Exp. 1 the mature seed contained 63% of the total plant 35S content at maturity. Because seeds had not yet begun to grow at the time of labeling, the entire quantity of label found in the seed had been mobilized from vegetative tissues. Seeds in Exp. 2 slowed their radiosulfur acquisition, taking up only a small amount of 35S after the fifth harvest. Seeds in this experiment acquired 18% of their total radiosulfur as 35SO4 and retained this portion through maturity. Seeds in Exp. 1 maintained approximately 7.5% of their 35S in the inorganic form.
Pulse 2
All tissues mobilized total-35S, 35SO4, and organic-35S (Fig. 3B, 3D, 3F, 4B, 4D, 4F) that was acquired at the beginning of rapid seed growth (R6.0 and R5.5, in Exp. 1 and 2, respectively) with efficiencies similar to those of 35S acquired in Pulse 2 (R4.0 and R2.0, in Exp. 1 and 2, respectively). Roots tended to mobilize this later acquired 35S slightly more efficiently in Exp. 1 than 35S taken up at R4.0, because of a much larger loss of 35SO4 from this later pulse. Because of a high uptake and retention of 35S in roots, and a limited initial labeling of leaves in Pulse 2 of Exp. 1 (Fig. 3B), roots mobilized nearly two times the labeled-S as did the leaves, although their mobilization efficiency was not as great. Roots lost large amounts of 35SO4 in Exp. 2 (Fig. 4D), allowing them to seem to be a more important source of S than leaves. Plants pulsed later appeared to reduce a larger portion of their total plant label after the first harvest (17 and 25% in Exp. 1 and 2, respectively), and left more unreduced-35S in their tissues at maturity than plants taking up S before seed development.
While acquiring large amounts of mobilized S, seeds maintained a constant portion of their label as 35SO4 through harvest. Mature seeds contained 11 and 18% of their label as 35SO4 in Pulse 2 of Exp. 1 and 2, respectively. Again, these values compare with 7.6 and 18% found as 35SO4 in Pulse 1 of Exp. 1 and 2.
Additional Pulses
Approximately half (44 to 63%) of the mature plants' label was mobilized to the seed after each of the seven individual pulses in the two experiments (Table 1). In Exp. 2, this value tended to increase through the first three pulses; however, the inverse occurred in the first experiment. The low mobilization rate found in plants supplied with 35SO4 in Pulse 2 (at DAI 79) of Exp. 1 was probably due to the longer pulse time used in this experiment.
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Mobilization efficiencies of the leaf fraction, and the relative sink strength for labeled S by the seed during growth can be seen in Fig. 5. The difference between initial 35S (open symbols) and final 35S (closed symbols) gives an indication of the relative strength of a tissue as a source or as a sink for S acquired at five distinct reproductive stages (R2.0 to R7.0). The pod fraction at maturity was not shown for clarity. The pods contained 4 ± 0.5% of the mature plants' 35S across all five pulses.
Sulfur Uptake and Mobilization Patterns of Expanding Leaves
Expanding leaves were harvested separately from the mature leaf fraction in Exp. 2 to examine intra-leaf fractional S uptake and mobilization patterns. Three newly opened leaves and any leaf buds were harvested as a fraction discrete from the mature leaves in the five harvests before maturity. Because of the indeterminate nature of the plants used in this study, mature leaf fractions of harvests at 81, 98, and 107 DAI contained leaves that were in the expanding leaf fraction at the time of the previous harvest. Conversely, the expanding leaf fraction from these harvests primarily contained leaves formed de novo since the previous harvest.
The expanding leaf fraction took up 44% of the total leaf label (Fig. 6A) while only contributing 19% to the entire leaf fraction's dry weight (data not shown) in plants pulsed at 65 DAI. While the expanding leaf fraction contained nearly four times the 35S concentration as the mature leaf fraction, they contained ten times the 35SO4 concentration (data not shown). The mature leaf fraction increased in total 35S substantially between the first and second harvests of the first pulse, because leaves that were formerly in the expanding leaf fraction became integrated into the mature leaf group. By the second harvest of plants pulsed at 65 DAI, the 35S content of the expanding leaf tissue dropped to near zero. Only 1 to 4% of the total leaf fraction's 35S could be found in subsequently harvested new leaves.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Plants in our study accumulated 56 to 59% of their total plant-S in leaves late in vegetative development. Through mobilization, leaves supplied the seed with 20% of its total S requirement. Pods, also important in this regard, contributed 10% of seed-S content. Although plants had a continuous supply of SO4 from the nutrient solution at their disposal through late seed growth, 40 and 36% (Exp. 1 and 2, respectively) of the developing seeds' S needs was met by mobilized S. Sunarpi and Anderson's (1997b) S-stressed soybeans fulfilled only 13% of the seeds' S need though mobilization. The contrasting results seem to demonstrate the importance of S availability before reproductive development for storage. It appears that soybean will mobilize previously acquired S to seed, if S is supplied in adequate quantities before seed filling. This emphasizes the importance of S reserve capacity in vegetative tissues.
Radiosulfur delivered to the plant in pulses during reproductive development was more efficiently mobilized to seed than S acquired throughout development. Whereas only 15% of mature plant total S appeared to have been mobilized from vegetative tissues in Exp. 2, 44 to 60% of the 35S found in mature plants was mobilized to seed from vegetative tissues following a 10 h pulse. All vegetative tissues lost 35SO4 (through reduction and/or mobilization) and mobilized organic 35S. Leaves were the major suppliers of mobilized 35S that was acquired by the plant early in reproductive development, but contributed a smaller portion of the mobilized vegetative 35S by the time of rapid seed growth. Differences in total leaf 35S mobilized were due mainly to a smaller 35S acquisition by the leaves, since their mobilization efficiency changed little through seed fill.
Sulfur taken up later in seed development was more likely to be mobilized to the seed. Plants pulsed after R5.0 mobilized a larger portion of their total label to seeds than did those pulsed before rapid seed growth (Fig. 3). In our study, changes in overall whole-plant mobilization efficiency over time of S acquisition did not seem to be caused by an increase in leaf mobilization efficiency. In fact, leaves mobilized 35S acquired in three pulses between R2.0 and R5.5 at 50% efficiency (Table 1). Leaf tissue retained a slightly smaller portion of the 35S administered in each of these three 10 h pulses, while the pod fraction increased. Because vegetative growth slows during rapid seed growth, S acquired by leaves later in development may be quickly exported to pods and seed without entering the leaf protein fraction.
This study of soybean in its reproductive phase supports and extends the work of others (Smith and Lang, 1988; Bell et al., 1995; Sunarpi and Anderson, 1997a; 1998) by noting that expanding soybean leaves acquire a disproportionately large amount of 35S relative to their contribution to the entire leaf fraction dry weight. Leaves that are near full expansion have been shown to take up S readily and quickly mobilize it to younger developing leaves (Smith and Lang, 1988; Sunarpi and Anderson, 1996a; 1997a, 1998). Ninety percent of this S was found to be organic S (Smith and Lang, 1988). Expanding leaves in this study contained three to four times the 35S concentration as mature leaves directly after each of four pulses through R6.5, and these new leaves contained a larger fraction of this label as SO4 than was seen in the mature leaf fraction. Although no direct evidence was uncovered, it appears that mature leaves quickly transfer newly acquired SO4 to developing leaves. Sulfur acquired by the new leaves during the 10 h pulse appears to be utilized there and not redistributed, because new leaf fractions taken at subsequent harvests contained almost zero 35S. This is consistent with the quick and transient redistribution of newly acquired SO4 via a xylem-to-phloem transfer, as suggested by Sunarpi and Anderson (1997a).
Sulfate in root and stem tissues was unavailable to expanding leaves. These tissues of plants pulsed at R2.0 housed 300 to 400 kBq 35SO4; however, this SO4 was not incorporated into new leaves formed in the chase period between the first and second harvests. Although the new leaf fraction of plants pulsed at R4.0 took up a high concentration of 35S, plants pulsed at R2.0 supplied expanding leaves solely with unlabeled S up to R4.0. Previously acquired 35S seemed unavailable to newly expanding leaves. It appears that new leaves are dependent on newly acquired S, and that S taken up earlier may enter a discrete pool that is largely unavailable to expanding leaves.
Within the 10 h pulses (Exp. 2), root and stem tissues accumulated significant quantities of reduced 35S. Because SO4 has been shown to be principally transported to expanding leaves for reduction (Anderson, 1990; Brunold, 1990), it is probable that leaves contributed extensively to the large and rapid accumulation of organic 35S in these tissues. Root acquisition of S mobilized from the shoot via phloem has been noted (Cooper and Clarkson, 1989; Herschbach and Rennenberg, 1995), while root reduction seems to be a minor component of whole-plant reduction (Anderson, 1990; Sexton and Shibles, 1999). Though mechanistically difficult to interpret, it appears that mature leaves may be quickly and transiently transferring newly acquired 35SO4 to expanding leaves, while reducing 35SO4 for export to root. Sunarpi and Anderson (1998) showed the root to be involved in the recycling of newly acquired S between fully expanded leaves and newly developing ones. Forms of S were not identified in their study. Regardless of its origin, root organic 35S was later mobilized in this study.
Very young developing pods took up disproportionately large quantities of 35S relative to their dry weight in Exp. 1. Pods received 35S at a far greater rate than could be supported by transpiration, indicating xylem-to-phloem transfer (Sunarpi and Anderson, 1997b). Pods were just initiating development at the start of Exp. 2; therefore, nearly all of their 35S was acquired indirectly during the chase, from roots, stems, or leaves. In both experiments and pulses, pods began with, and maintained, a much larger portion of their label as 35SO4 than leaves. Although they did not appear to store great amounts of 35S, we found pods to be efficient mobilizers of total S, as previously noted (Sexton et al., 1998b; Anderson and Fitzgerald, 2001). Although S compounds were not examined, Thorne (1979) found soybean pods to be important for storage and mobilization of starch, reducing sugars, and nitrogenous materials. Pods certainly play a role in transferring S to seed and may be important in reducing SO4 for mobilization to developing seed as proposed by Sunarpi and Anderson (1997b).
Seeds acquired little label within the 10-h pulse used in Exp. 2 (Fig. 2B); however, pods acted as active sinks for newly acquired S. Seeds did acquire significant amounts of newly acquired S within a few days, however. As with Sunarpi and Anderson (1997b), it is impossible to determine from this study whether seed was taking up organic or inorganic S through the pods. Sexton and Shibles (1999) quantified the contribution of individual plant organs toward total plant ATP sulfurylase activity in soybean during reproductive development. Approximately 50% of total shoot's ATP sulfurylase activity was found in the seeds, while leaves contributed only about 30 to 40% during rapid seed filling. Stems and pods each provided approximately 10% of the plant's total ATP sulfurylase activity (Sexton and Shibles, 1999). Also, soybean cotyledons grown in vitro are able to reduce SO4. Soybean cotyledons grown in a nutrient medium with SO4 as a sole S source accumulate storage proteins containing significant quantities of methionine (Holowach et al., 1984). Although support for S reduction occurring in seeds is likely (Sexton et al., 2002), our study does not exclude the possibility that leaves and/or pods are reducing and exporting S obtained as SO4 to seed.
Our study does show that SO4 made up only a small fraction of the leaves' total 35S content, even after allowing just 10 h for reduction. This indicates that SO4 reduction or export was occurring rapidly in leaf tissue. Pods, on the other hand, seemed to acquire large amounts of their total label as SO4 and reduced or mobilized it to seed throughout seed growth. Sunarpi and Anderson (1997b) thought that the large loss of pod SO4 early in seed development was because of reduction and mobilization of organic S to seed, because of the presence of a very low seed-SO4 concentration. Because their plants were S stressed, it is possible that pods passed along SO4 to the seed, where it was quickly reduced. Again, flux is difficult to estimate given only a steady state "snapshot" of 35S levels. In our study, seeds continued to acquire significant quantities of 35SO4 throughout development independent of pulse date or experiment, indicating that seeds have the ability to take up and accumulate SO4. Whether this implies that a significant amount of SO4 reduction was occurring within the seed is conjectural.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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In this study, seeds acquired and maintained a large fraction of their 35S pool as SO4. Seeds of plants pulsed at R2.0 and supplied with sufficient N and S retained approximately 18% of their 35S in the 35SO4 fraction. This implies that an expansion of the seed SO4 reductive capacity late in seed development may help to increase soybean seed protein quality.
Soybean leaves have been shown repeatedly to be the primary donors of N for mobilization to seed tissue (Zeiher et al., 1982; Loberg et al., 1984; Egli and Leggett, 1985; Shibles and Sundberg, 1998). We have demonstrated their importance as providers of reduced S as well. Under SO4 sufficient conditions, leaves supplied the seed with 20% of its total-S requirement and up to 80% of the mobilized S that was obtained as a single pulse of 35S during reproductive development. Similar to N (Pate and Flinn, 1973; Loberg et al., 1984) the quantity of S mobilized from leaves appears to be most reliant on the quantity stored.
The generation of transgenic soybean overexpressing methionine- and cysteine-rich leaf proteins would be particularly valuable in studying S mobilization. A vegetative storage protein for S storage would be best expressed early in leaf development, as we found expanding leaves to accumulate 35S at high rates. These transformants could supply the seed with reduced-S during senescence, when developing seed appear to be starved for methionine and cysteine.
Received for publication February 21, 2005.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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' and ß subunit genes in transgenic plants. Plant Mol. Biol. 11:109–123.[CrossRef][ISI]This article has been cited by other articles:
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  S. L. Naeve, T. A. O'Neill, and J. E. Miller-Garvin Canopy Nitrogen Reserves: Impact on Soybean Yield and Seed Quality Traits in Northern Latitudes Agron. J., May 7, 2008; 100(3): 681 - 689. [Abstract] [Full Text] [PDF]
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