Kinetics of C-14 Translocation in Soybean: II. Kinetics in the Leaf

The kinetics of ¹⁴C-assimilates in the soybean leaf were studied in pulse labeling and steady state labeling experiments. ¹⁴C-Sucrose apparently served as the ultimate source, at least, of translocated ¹⁴C-sucrose. However, since the specific activity of leaf sucrose reached a maximum within 5 minutes after pulse labeling, whereas that of exported sucrose did not reach a maximum until at least 20 minutes, it appeared that there were two sucrose compartments in the leaf. A possible physical basis for the two compartments may be the mesophyll (a photosynthetic compartment) and a specialized “paraveinal mesophyll” (a nonphotosynthetic compartment), through which photosynthate must pass on its way to the veins.     

Translocation of C Sucrose in Sugar Beet during Darkness

The time-course of arrival of ¹⁴C translocate in a sink leaf was studied in sugar beet (Beta vulgaris L. cultivar Klein Wanzleben) for up to 480 minutes of darkness. Following darkening of the source leaf, translocation rapidly declined, reaching a rate approximately 25% of the light period rate by 150 minutes. Comparison of data from plants that were girdled 1 cm below the crown with data from ungirdled plants indicates that after about 150 minutes darkness the beet root becomes a source of translocate to the sink leaf. After about 90 minutes darkness, starch-like reserve polysaccharide from the source leaf begins to contribute ¹⁴C to ethanol soluble pools in that leaf. Because of a 15% isotope mass effect, sucrose, at isotopic saturation, reaches a specific activity which is about 85% of the level of the supplied CO₂. The source leaf sucrose specific activity remains at the isotopic saturation level for about 150 minutes of darkness, after which time input from polysaccharide reserves causes the specific activity to drop to about 55% of that of the supplied CO₂. Sucrose specific activity determinations, polysaccharide dissolution measurements, and pulse labeling experiments indicate that following partial depletion of the sucrose pool, source leaf polysaccharide contributes to dark translocation. Respired CO₂ from the source leaf appears to be derived from a pool which, unlike sucrose, remains at a uniform specific activity.     

Kinetics of C-photosynthate translocation in morning glory vines

The movement of ¹⁴C-photosynthate in morning glory (Ipomea nil Roth, cu. Scarlet O'Hara) vines 2 to 5 meters long was followed by labeling a lone mature leaf with ¹⁴CO₂ and monitoring the arrival rate of tracer at expanding sink leaves on branches along the stem. To a first approximation, the kinetic behavior of the translocation profiles resembled that which would be expected from movement at a single velocity (“plug flow”) without tracer loss from the translocation stream. There was no consistent indication of a velocity gradient along the vine length. The profile moved along the vine as a distinct asymmetrical peak which changes shape only slowly. The spatial distribution of tracer along the vine reasonably matched that predicted on the basis of the arrival kinetics at a sink, assuming plug flow with no tracer loss. These observations are in marked contrast to the kinetic behavior of any mechanism describable by diffusion equations.

However, a progressive change in profile shape (a symmetrical widening) was observed, indicating a range of translocation velocities. A minimum of at least two factors must have contributed to the observed velocity gradient: the exchange of ¹⁴C between sieve elements and companion cells (demonstrated by microautoradiography) and the range of velocities in the several hundred sieve tubes which carried the translocation stream. Possible effects of these two factors on profile spreading were investigated by means of numerical models. The models are necessarily incomplete, due principally to uncertainties about the exchange rate between sieve elements and companion cells and the degree of functional connectivity between sieve tubes of different conductivities. However, most of the observed profile spreading may be reasonably attributed to the combined effects of those two factors.

The mass average velocity of translocation (calculated from the mean times of ¹⁴C arrival at successive sink leaves) was about 75% of the maximum velocity (calculated from the times of initial detection at the same sink leaves), which was usually between 0.6 and 1 cm min⁻¹. Owing to tracer exchange between sieve elements and companion cells, the mass average velocity of tracer in the sieve tubes was probably closer to 86% of the maximum velocity, a figure which agreed with a predicted velocity distribution based on calculated sieve tube conductivities and the size distribution of functional sieve tubes.

     

Compartmentation in Vicia faba Leaves: II. Kinetics of C-Sucrose Redistribution among Individual Tissues following Pulse Labeling

Leaflets of Vicia faba L. were pulse labeled with ¹⁴CO₂ and the kinetics of ¹⁴C-sucrose redistribution among individual tissues was followed. Sucrose specific activity in the whole leaf peaked about 15 minutes after labeling and declined with a half-time of about 80 minutes. In one experiment, leaflet discs taken at various times during the ¹²CO₂ chase were quick frozen, freeze-substituted, and embedded in plastic. The tissue was sectioned paradermally and sections of palisade parenchyma, of spongy parenchyma, and of spongy parenchyma that contained veins were collected. Water extracts from these sections were assayed for sucrose specific activity. Sucrose specific activity in the palisade parenchyma was higher than that of the spongy parenchyma and reached a maximum in both tissues 9 to 15 minutes after labeling. Sucrose specific activity initially declined rapidly in the palisade parenchyma followed by a period during which little or no loss occurred. Sucrose specific activity in sections containing veins peaked at 15 minutes with a maximum value substantially higher than either mesophyll tissue, indicating that recently synthesized sucrose was preferentially exported from the mesophyll. Decline of activity in these sections containing veins continued for the remainder of the experiment. Sucrose specific activity in lower epidermal peels peaked several minutes after that of the whole leaflet and remained lower. Sucrose specific activity in upper epidermal peels was variable (probably due to contamination), but the limited data suggest that the sucrose specific activity there reached somewhat higher values than those of the lower epidermis. The experiments indicate that each leaf tissue contains a kinetically identifiable sucrose pool (which we refer to as “histological compartmentation”), and that further compartmentation may occur at the intracellular level. A simulation of leaf sucrose compartmentation is presented.     

Translocation and accumulation of translocate in the sugar beet petiole

Accumulation of translocate during steady-state labeling of photosynthate was measured in the source leaf petioles of sugar beet (Beta vulgaris L. monogerm hybrid). During an 8-hr period, 2.7% of the translocate or 0.38 μg carbon/min was accumulated per cm petiole. Material was stored mainly as sucrose and as compounds insoluble in 80% ethanol. The minimum peak velocity of translocation approached an average of 54 cm/hr as the specific activity of the ¹⁴CO₂ pulse was progressively increased. The ratio of cross sectional area required for translocation to actual sieve tube area in the petiole was 1.2. A regression analysis of translocation rate versus sieve tube cross sectional area yielded a coefficient of 0.76. The specific mass transfer rate in the petiole was 1.4 g/hr cm² phloem or 4.8 g/hr cm² sieve tube. Histoautoradiographic studies indicated that translocation occurs through the area of phloem occupied by sieve tubes and companion cells while storage occurs in these cells plus cambium and phloem parenchyma cells. The ability of the petiole to act as a sink for translocate is consistent with the concept that storage along path tissue serves to buffer sucrose concentration in the translocate during periods of fluctuating assimilation.     

Relationship between Photosynthesis and Protein Synthesis in Maize: I. Kinetics of Translocation of the Photoassimilated Carbon from the Ear Leaf to the Seed

To gain a better understanding of the biochemical basis for partitioning of photosynthetically fixed carbon between leaf and grain, a ¹⁴CO₂ labeling study was conducted with field-grown maize plants 4 weeks after flowering. The carbon flow was monitored by separation and identification of ¹⁴C assimilates and ¹⁴C storage components within each tissue during the chase period (from 4 to 96 hours) following a 5 minute ¹⁴CO₂ pulse. In the labeled ear leaf, the radioactivity strongly decreased to reach, at the end of the experiment, about 12% of the total incorporated radioactivity, mostly associated with sucrose and proteins. Nevertheless, an unexpected reincorporation of radioactivity was observed either in leaf starch or proteins, the day following the pulse. Conversely, the radioactivity in the grain increased to attain 66% of the total incorporated ¹⁴C after a 96 hour chase. The photosynthates, mostly sucrose, organic and free amino acids, rapidly translocated towards the developing seeds, served as precursors for the synthesis of seed storage compounds, starch, and proteins. They accumulate in free form for 24 hours before being incorporated within polymerized storage components. This delay is interpreted as a necessary prerequisite for interconversions prior to the polycondensations. In the grain, the labeling of the storage molecules, either in starch or in storage protein groups (salt-soluble proteins, zein, and glutelin subgroups), was independent of their chemical nature but dependent on their pool size.     

Source pool kinetics for C-photosynthate translocation in morning glory and soybean

The kinetic behavior of translocation profiles indicates that their shape is determined largely by the rate at which tracer enters the sieve tubes in the source leaf. Confirmation of this relationship was sought by investigating the kinetics of ¹⁴C in the immediate source pool for translocated sucrose in soybean (Glycine max L., cv. Bragg) and morning glory (Ipomea nil Roth, cv. Scarlet O'Hara) leaves. Quantitative microautoradiography was used to follow the water-soluble ¹⁴C contents of the companion cells in minor veins after pulse-labeling with ¹⁴CO₂. In both morning glory and soybean, the observed kinetics in the companion cells matched reasonably well those expected from the shape of the translocation profiles.

Marked compartmentation of sucrose was evident in soybean leaves in that the specific radioactivity of total leaf sucrose was greatest immediately after labeling and quickly declined, whereas labeling in the companion cells was low at first and did not reach a maximum for about 35 minutes. In morning glory leaves, the kinetics of sucrose specific radioactivity and of companion cell-labeling more closely paralleled one another.

     

Allocation and Turnover of Photosynthetically Assimilated CO(2) in Leaves of Glycine max L. Clark

The allocation and turnover of photosynthetically assimilated ¹⁴CO₂ in lipid and protein fractions of soybean (Glycine max L. Clark) leaves and stem materials was measured. In whole plant labeling experiments, allocation of photosynthate from a pulse of ¹⁴CO₂ into polymeric compounds was: 25% to proteins in 4 days, 20% to metabolically inert cell wall products in 1 to 2 days, 10% to lipids in 4 days, and 4% to starch in 1 day. The amount of ¹⁴C labeled photosynthate that an actively growing leaf (leaf 4) used for its own lipid synthesis immediately following pulse labeling was about 25%. The ¹⁴C of labeled proteins turned over with half-lives of 3.8, 3.3, and 4.1 days in leaves 1, 2, and 3, respectively; and turnover of ¹⁴C in total shoot protein proceeded with a half-life of 5.2 days. Three kinetic ¹⁴C turnover patterns were observed in lipids: a rapid turnover fraction (within a day), an intermediate fraction (half-life about 5 days), and a slow turnover fraction. These results are discussed in terms of previously published accounts of translocation, carbon budgets, carbon use, and turnover in starch, lipid, protein, and cell wall materials of various plants including soybeans.     

Sucrose Transport and Phloem Unloading in Stem of Vicia faba: Possible Involvement of a Sucrose Carrier and Osmotic Regulation

Stems of Vicia faba plants were used to study phloem unloading because they are hollow and have a simple anatomical structure that facilitates access to the unloading site. After pulse labeling of a source leaf with ¹⁴CO₂, stem sections were cut and the efflux characteristics of ¹⁴C-labeled sugars into various buffered solutions were determined. Radiolabeled sucrose was shown to remain localized in the phloem and adjacent phloem parenchyma tissues after a 2-hour chase. Therefore, sucrose leakage from stem segments prepared following a 75-minute chase period was assumed to be characteristic of phloem unloading. The efflux of ¹⁴C assimilates from the phloem was enhanced by 1 millimolar p-chloromercuribenzene sulfonic acid (PCMBS) and by 5 micromolar carbonyl cyanide m-chlorophenly hydrazone (CCCP). However, PCMBS inhibited and CCCP enhanced general leakage of nonradioactive sugars from the stem segments. Sucrose at concentrations of 50 millimolar in the free space increased efflux of [¹⁴C]sucrose, presumably through an exchange mechanism. This exchange was inhibited by PCMBS and abolished by 0.2 molar mannitol. Increasing the osmotic concentration of the efflux medium with mannitol reduced [¹⁴C]sucrose efflux. However, this inhibition seems not to be specific to sucrose unloading since leakage of total sugars, nonlabeled sucrose, glucose, and amino acids from the bulk of the tissue was reduced in a similar manner. The data suggest that phloem unloading in cut stem segments is consistent with passive efflux of sucrose from the phloem to the apoplast and that sucrose exchange via a membrane carrier may be involved. This is consistent with the known conductive function of the stem tissues, and contrasts with the apparent nature and function of unloading in developing seeds.     

The influence of externally supplied sucrose on phloem transport in the maize leaf strip

Sucrose (2,5–1000 mmol l^–1), labeled with [¹⁴C]sucrose, was taken up by the xylem when supplied to one end of a 30-cm-long leaf strip of Zea mays L. cv. Prior. The sugar was loaded into the phloem and transported to the opposite end, which was immersed in diluted Hoagland's nutrient solution. When the Hoagland's solution at the opposite end was replaced by unlabeled sucrose solution of the same molarity as the labeled one, the two solutions met near the middle of the leaf strip, as indicated by radioautographs. In the dark, translocation of ¹⁴C-labeled assimilates was always directed away from the site of sucrose application, its distance depending on sugar concentration and translocation time. When sucrose was applied to both ends of the leaf strip, translocation of ¹⁴C-labeled assimilates was directed toward the lower sugar concentration. In the light, transport of ¹⁴-C-labeled assimilates can be directed (1) toward the morphological base of the leaf strip only (light effect), (2) toward the base and away from the site of sucrose application (light and sucrose effect), or (3) away from the site of sucrose application independent of the (basipetal or acropetal) direction (sucrose effect). The strength of a sink, represented by the darkened half of a leaf strip, can be reduced by applying sucrose (at least 25 mmol l^–1) to the darkened end of the leaf strip. However, equimolar sucrose solutions applied to both ends do not affect the strength of the dark sink. Only above 75 mmol l^–1 sucrose was the sink effect of the darnened part of the leaf strip reduced. Presumably, increasing the sucrose concentration replenishes the leaf tissue more rapidly, and photosynthates from the illuminated part of the leaf strip are imported to a lesser extent by the dark sink.Supported by Deutsche Forschungsgemeinschaft     

Influence of Translocation of Photosynthetic Efficiency of Phaseolus vulgaris L

Measurements of net photosynthesis show that in Phaseolus vulgaris L. the cultivar Michelite-62 exceeds the cultivar Red Kidney in net CO₂ uptake by 23 to 31%. Data on translocation of pulse label indicate that export of a pulse of photosynthetically assimilated ¹⁴C from the source leaf of either M-62 or Red Kidney follows an exponential pattern and shows an initial rapid phase followed by a second slower phase. The steeper slope for both phases in M-62 suggests its rate of translocation of pulse label is higher than that of Red Kidney. Furthermore, only 38% of the ¹⁴C remains in the leaf of M-62 after 8 hours, while Red Kidney retains up to 60% of the label. Leaf autoradiographs obtained after pulse labeling demonstrate a much faster rate of vein loading in M-62 and are considered evidence for the higher translocation efficiency of M-62. These results provide evidence for a positive correlation between photosynthetic efficiency and translocation efficiency in M-62 and Red Kidney and give support to our hypothesis that translocation is one of the important physiological factors controlling the varietal differences in photosynthetic efficiency in Phaseolus vulgaris.     

Quantitative analysis of transpiration stream dynamics in an intact cucumber stem by a heat flux control method

Water flux of transpiration stream in an intact stem of the 10 leaf stage cucumber plant (Cucumis sativus L. cv. Chojitsu-Ochiai) was measured by a novel system of heat flux control method with a resolution of 1 × 10⁻³ grams per second and a time constant of 1 minute; two heat flux control sensors were attached to the seventh internode and the stem base. The transpiration stream responded clearly to leaf transpiration and root water absorption when the plant was exposed to light, and the water flux at the stem base corresponded to the transpiration rate per plant in steady state. Root water absorption lagged about 10 minutes behind leaf transpiration. Dynamics of water fluxes were affected by the lag of water absorption in roots, and temporary water loss caused by rapid increase in leaf transpiration was buffered by about 5% of the water content in the stem.     

Effect of Moisture Supply upon Translocation and Storage of C in Sugarcane

Low moisture supply, controlled by 3 methods (adding NaCl to a complete nutrient solution, allowing a cut stalk to wilt, or withholding irrigation in the field), decreased the velocity and percentage rate of translocation of ¹⁴C-photosynthate. The surplus sucrose not used in growth moved more slowly in the phloem and was stored in the stalk.

Low moisture supply depressed translocation of ¹⁴C-photosynthate more severely than it curtailed formation of ¹⁴C-photosynthate in the same leaf: therefore, the effect of moisture supply upon translocation was primary.

Low moisture supply retarded profile development in the stem, and a loss in moisture gradient was associated with a steepened slope of the profile. These results indicate a flow mechanism of translocation rather than diffusion.

Results reported now and previously point to the operation of a slow pressure-flow mechanism particularly during the night but also during the day; superimposed upon this general mass transport is the more rapid process of phototranslocation which is independent of sugar gradients and which can cause the accumulation of sucrose at the storage-sink.

During ripening, storage of sucrose in the stalk may be increased by withholding water because less sucrose is hydrolyzed in transit, less is used in growth, and the slowly moving sucrose has more time for transfer from the phloem to the storage parenchyma.

     

Synthesis and Storage of Sucrose in Relation to Activities of Its Metabolizing Enzymes in Sugarcane Cultivars Differing in Maturity

Metabolic changes in the contents of sucrose and hexoses in relation to the activities of invertase, sucrose synthase and sucrose-phosphate synthase in early (CoJ 64) and late (Co 1148) maturing cultivars of sugarcane have been studied. During early stages of cane growth, lower activities of sucrose synthase and sucrose-phosphate synthase in leaf blade In CoJ 64 over Co 1148 were observed. However, sucrose content in sheath/blade was higher in CoJ 64 than in Co 1148. With the advancing age, the activity of soluble acid invertase (pH 5.4) in stem declined more rapidly in CoJ 64. This resulted in building up of high ratio of sucroselinvert sugars in stem tissue of this cultivar. Feeding uniformly-labelled sucrose and glucose to the cut discs of leaf sheath resulted in higher uptake of ¹⁴C in CoJ 64 than in Co 1148. Uptake by stem tissue discs of ¹⁴C from sucrose was less than that from hexoses. Based on these results, it is suggested that (i) the rapid fall in the activity of soluble acid invertase in stem concomitant with fast accumulation of sucrose in this tissue is an index of early maturity of the cane, and (ii) high content of sucrose in sheath is a reflection of an efficient translocation of this sugar in early maturing cultivars.     

Phloem transport of 14C-labelled assimilates in Ricinus

Summary Exudate can be obtained from incisions made in the bark of the stem of actively growing Ricinus plants. ¹⁴C-labelled assimilates from a fed leaf are rapidly detected in the exudate. This movement was both acropetal and basipetal from the fed leaf, at rates of over 100 cm h^-1. Estimated rates within intact plants were 80–84 cm h^-1.In contrast with xylem sap obtained from the same plant, the exudate obtained had an alkaline pH (8.2), a high dry matter content (10–12.5%), high sugar content (8–10%) which was predominantly sucrose; high potassium content (60–80 mM) and low calcium content (0.5–1.0 mM).It is concluded, on the basis of the present evidence, that the exudate is a true sample of the sieve tube sap undergoing translocation.     

Source and sink leaf metabolism in relation to Phloem translocation: carbon partitioning and enzymology

The import-export transition in sugar beet leaves (Beta vulgaris) occurred at 40 to 50% leaf expansion and was characterized by loss in assimilate import and increase in photosynthesis. The metabolism and partitioning of assimilated and translocated C were determined during leaf development and related to the translocation status of the leaf. The import stage was characterized by C derived from either ¹⁴C-translocate or ¹⁴C-photosynthate being incorporated into protein and structural carbohydrates. Marked changes in the C partitioning were temporally correlated with the import-export conversion. Exporting leaves did not hydrolyze accumulated sucrose and the C derived from CO₂ fixation was preferentially incorporated into sucrose. Both source and sink leaves contained similar levels of acid invertase and sucrose synthetase activities (sucrose hydrolysis) while sucrose phosphate synthetase (sucrose synthesis) was detected only in exporting leaves. The results are discussed in terms of intracellular compartmentation of sucrose and sucrose-metabolizing enzymes in source and sink leaves.     

Enhancement of [C]Sucrose Export from Source Leaves of Vicia faba by Gibberellic Acid

The effect of gibberellic acid (GA₃) on sucrose export from source leaves was studied in broad bean (Vicia faba L.) plants trimmed of all but one source and one sink leaf. GA₃ (10 micromolar) applied to the source leaf, enhanced export of [¹⁴C]sucrose (generated by ¹⁴CO₂ fixation) to the root and to the sink leaf. Enhanced export was observed with GA treatments as short as 35 minutes. When GA₃ was applied 24 hours prior to the ¹⁴CO₂ pulse, the enhancement of sucrose transport toward the root was abolished but transport toward the upper sink leaf was unchanged. The enhanced sucrose export was not due to increased photosynthetic rate or to changes in the starch/sucrose ratio within the source leaf; rather, GA₃ increased the proportion of sucrose exported. After a 10-min exposure to [¹⁴C]GA₃, radioactivity was found only in the source leaf. Following a 2 hour exposure to [¹⁴C]GA₃, radioactivity was distributed along the entire stem and was present in both the roots and sink leaf. Extraction and partitioning of GA metabolites by thin layer chromatography indicated that there was a decline in [¹⁴C]GA₃ in the lower stem and root, but not in the upper stem. This pattern of metabolism is consistent with the disappearance of the GA₃ effect in the lower stem with time after treatment. We conclude that in the short term, GA₃ enhances assimilate export from source leaves by increasing phloem loading. In the long term (24 hours), the effect of GA₃ is outside the source leaf. GA₃ accumulates in the apical region resulting in enhanced growth and thus greater sink strength. Conversely, GA₃ is rapidly metabolized in the lower stem thus attenuating any GA effect.     

Comparison of upward and downward translocation of C from a single leaf of sunflower

When single leaves attached at a given node were allowed to carry on photosynthesis in ¹⁴CO₂ for 30 min, younger plants showed a higher proportion of upward translocation than did older plants. Downward translocation of ¹⁴C-photosynthate was stimulated by ATP pre-treatment of the translocating leaf, while upward translocation was not affected by ATP. A similar phenomenon was observed in the translocation of ¹⁴C-sucrose infiltrated into a leaf with or without ATP. Downward translocation of photosynthate was inhibited by DNP pre-treatment of a fed leaf. Upward translocation, however, was not affected by DNP. Thirty min after infiltration of ¹⁴C-glucose into a leaf, almost all the ¹⁴C translocated upwards was found to be in the form of glucose, while a great part of the ¹⁴C translocated downwards was in the form of sucrose. In the case of translocation of infiltrated ¹⁴C-sucrose, ¹⁴C found both above and below the fed leaf was mainly in the form of sucrose.     

Evidence for thiol-induced enhanced in situ translocation of 14C-sucrose from source to sink in Brassica juncea

The translocation of ¹⁴C-sucrose to the different parts of the mustard (Brassica juncea) crop has been evaluated in the context of understanding the source to sink relationship in the thiol-induced enhanced crop yield. The foliar application of thiols like TU, TGA and DTT to the plant gave maximum sucrose phosphate synthase activity, which was found to have direct correlation with the movement of sucrose. The distribution pattern of ¹⁴C-sucrose follows the path from internode and node to pod via leaf. The translocation of ¹⁴C-sucrose was found to be a light dependent process. Among the nucleotides ATP and GTP, only ATP was able to promote the translocation and GTP was ineffective. In this unique in situ tracer experiment using ¹⁴C-sucrose, we could establish that thiols are able to enhance the translocation of sucrose from source to sink.     

Evidence for thiol-induced enhanced in situ translocation of 14C-sucrose from source to sink in Brassica juncea

The translocation of ¹⁴C-sucrose to the different parts of the mustard (Brassica juncea) crop has been evaluated in the context of understanding the source to sink relationship in the thiol-induced enhanced crop yield. The foliar application of thiols like TU, TGA and DTT to the plant gave maximum sucrose phosphate synthase activity, which was found to have direct correlation with the movement of sucrose. The distribution pattern of ¹⁴C-sucrose follows the path from internode and node to pod via leaf. The translocation of ¹⁴C-sucrose was found to be a light dependent process. Among the nucleotides ATP and GTP, only ATP was able to promote the translocation and GTP was ineffective. In this unique in situ tracer experiment using ¹⁴C-sucrose, we could establish that thiols are able to enhance the translocation of sucrose from source to sink.