Reduction of the cytosolic fructose-1,6-bisphosphatase in transgenic potato plants limits photosynthetic sucrose biosynthesis with no impact on plant growth and tuber yield

Sucrose produced in source leaves is the predominant carbon source for developing sink tissues in most higher plants. Consequently the rate of sucrose synthesis is likely to be important for sink development and final crop yield. Two sucrose biosynthetic enzymes are believed to possess regulatory properties with respect to the rate of sucrose synthesis: (i) cytosolic FBPase and (ii) sucrose phosphate synthase. To study the impact of reduced photosynthetic sucrose biosynthesis on plant growth and crop yield a cDNA clone encoding cytosolic FBPase was isolated from a potato leaf cDNA library and used for antisense experiments in transgenic potato plants. The cDNA clone cy-F1, containing an open reading frame of 1020 bp highly homologous (85%) to other known sequences of plant cytosolic FBPases, was cloned in reversed orientation between the 35S CaMV promoter and the octopine synthase polyadenylation signal. Out of 75 independent transformants five transgenic lines having 9 to 55% of the wild-type FBPase activity were chosen for further analysis. A 45% reduction of the cytosolic FBPase activity did not cause any measurable change in metabolite concentrations, growth behaviour or photosynthetic parameters of the transgenic plants. Inhibition of cytosolic FBPase activity below 20% of the wild-type activity led to an accumulation of 3-PGA, triose-phosphates and fructose-1,6bisphosphate in source leaves. This resulted in a reduced light-saturated rate of assimilation measured via gas exchange and a decreased photosynthetic rate under conditions of the leaf disc electrode with saturating light and CO₂. Measuring photosynthetic carbon fluxes by labelling leaf discs with ¹⁴CO₂ revealed a 53–65% reduction of sucrose synthesis whereas starch synthesis decreased only by 18–24%. The flux into the anionic and cationic fraction was not altered. Despite these changes steadystate sucrose concentrations were not effected in source leaves from transgenic plants. Starch accumulated by more than a factor of 3 compared with wild-type leaves and was degraded during the night. This provides strong evidence for the hypothesis that hexoses and/or hexosephosphates are exported out of the chloroplasts, thereby circumventing the limitation of sucrose biosynthesis caused by the inhibition of cytosolic FBPase in the dark. Accordingly, plant growth and potato tuber yield remained unaltered. From these data it can be concluded that a reduced photosynthetic sucrose biosynthetic capacity can be efficiently compensated without any reduction in crop yield under greenhouse or growth chamber conditions by changing carbon export strategy. Whether the same holds true for field conditions remains to be elucidated.     

Sources of Carbon for Export from Spinach Leaves throughout the Day

Rates of net carbon exchange, export, starch, and sucrose synthesis were measured in leaves of spinach (Spinacia oleracea L.) throughout a 14-hour period of sinusoidal light to determine the sources of carbon contributing to export. Net carbon exchange rate closely followed light level, but export remained relatively constant throughout the day. In the morning when photosynthesis was low, starch degradation provided most of the carbon for export, while accumulated sucrose was exported during the evening. At high photosynthesis rate, the regulatory metabolite fructose 2,6-bisphosphate was low, allowing more of the newly fixed carbon to flow to sucrose through cytosolic fructose bisphosphatase. When the rate of sucrose synthesis exceeded the rate of export from the leaf, sucrose accumulated and soon thereafter sucrose synthesis declined. A decreasing sucrose synthesis rate resulted in additional carbon moving to the synthesis of starch, which was maintained throughout the remainder of the day. The declining sucrose synthesis rate coincided with decreasing activity of sucrose phosphate synthase present in gel-filtered leaf extracts. A rise in the leaf levels of uridine diphosphoglucose and fructose 6-phosphate throughout the day was consistent with this declining activity.     

The Effect of Nitrogen Supply to Urtica dioica L. Plants on the Distribution of Assimilate Between Shoot and Roots

In this study the influence of nitrogen nutrition on the patterns of carbon distribution was investigated with Urtica dioica. The nettles were grown in sand culture at 3 levels of NO^?₃, namely 3 (low), 15 (medium) and 22 (high) mM. These levels encompassed a range within which nitrogen did not affect total biomass production. The ratio of root: shoot biomass of the low nitrogen plants was, however, significantly higher than that of the nettles grown at medium and high N supply. Carbon allocation from one leaf of each pair of leaves was examined after a ¹⁴CO₂-pulse and a subsequent ¹⁴C distribution period of one night. Only the youngest two leaf pairs did not export assimilates. Carbon (¹⁴C) export to the shoot apex and to the roots, as measured at the individual nodes responded to the nitrogen status: At medium and high nitrogen supply the 3rd, 4th and 5th leaf pairs exported to the shoot apex, while lower leaves exported to the root. At low nitrogen supply only the 3rd leaf exported towards the shoot apex. The results illustrate the plastic response of carbon distribution patterns to the nitrogen supply, even when net photosynthesis, carbon export from the source leaves and biomass production were not affected by the nitrogen supply to the plant.     

Mechanism of export of organic material from the developing fruits of pea

Pisum sativum L. fruits export a small quantity of radiolabeled substances to other plant parts after the fruits are allowed to photosynthesize in the presence of ¹⁴CO₂. Export was uninhibited by peduncle girdling suggesting an apoplastic route for transport of material, presumably by `reverse' flow in the peduncle xylem. To determine if any diurnal water potential gradient formed between pea leaves and fruit might be responsible for the observed export, the water potentials of the various organs were monitored over 24 hours. Water potential differences of up to 7.5 bars existed between leaves and fruit in long photoperiods, and up to 2.5 bars in short photoperiods. Pulses of <sup>14</sup>CO<sub>2</sub> labeling indicated that initial delivery of exported label was to `transpirational sinks,' with subsequent redistribution of label to metabolic sinks. Export to the apical bud appeared to be direct via the xylem. Application of membrane-impermeable inulin to a surgically opened seed coat `cup' resulted in export mainly to the subtending leaf with little redistribution. Simultaneous application of sucrose to the seed coat resulted in more extensive distribution of the sucrose, consistent with reloading of the sucrose into mature leaf phloem. Thus, export of material from fruits appears to occur via a xylem pathway in response to transpirationally derived water potential gradients.     

Carbon partitioning and export from mature cotton leaves

The partitioning of carbon in intact, mature cotton (Gossypium hirsutum L.) leaves was examined by steady-state ¹⁴CO₂ labeling. Plants were exposed to dark periods of varying lengths, followed by similar illuminated labeling periods. These treatments produced leaves with a range of starch and soluble sugar contents, carbon exchange, and carbon export rates. Export during the illuminated periods was neither highly correlated with photosynthesis nor was export during the illuminated periods significantly different among the treatments. In contrast, the rate of subsequent nocturnal carbon export from these leaves varied widely and was found to be highly correlated with leaf starch content at the end of the illumination period (r = 0.934) and with nocturnal leaf respiration (r = 0.954). Leaves which had accumulated the highest levels of starch (about 275 micrograms per square centimeter) by the end of the illumination period exhibited nocturnal export rates very similar to those during the daylight hours. Leaves which accumulated starch to only 50 to 75 micrograms per square centimeter virtually ceased nocturnal carbon export. For leaves with starch accumulations of between 50 and 275 micrograms per square centimeter, nocturnal export was directly proportional to leaf starch at the end of the illumination period. After the nocturnal export rate was established, it continued at a constant rate throughout the night even though leaf starch and sucrose contents declined.     

Daytime and nighttime carbon balance and assimilate export in soybean leaves at different photon flux densities

To evaluate daytime and nighttime carbon balance and assimilate export in soybean (Glycine max [L.] Merrill) leaves at different photon flux densities, rates of CO₂ exchange, specific leaf weights, and concentrations of sucrose and starch were measured at intervals in leaves of pod-bearing `Amsoy 71' and `Wells II' plants grown in a controlled environment room. Assimilate export was estimated from CO₂ exchange and change in specific leaf weight. Total diurnal assimilate export was similar for both cultivars. Large cultivar differences existed, however, in the partitioning of carbon into starch reserves and the relative amounts of assimilate exported during the day and the night. Total amounts of both daytime and nighttime export increased with increasing photon flux density, as did sucrose and starch concentrations, specific leaf weight, and rate of respiratory carbohydrate loss at night. Cultivar differences in nighttime rate of export were more closely related to the differences in amount of assimilate available at the end of the day than to differences in daytime rate of net CO₂ assimilation. Daytime rates of export, however, were closely related to daytime rates of net CO₂ assimilation within each cultivar. The total amount of starch depleted during the 10-hour night increased as starch concentration at the beginning of the night increased.     

Glyphosate inhibits photosynthesis and allocation of carbon to starch in sugar beet leaves

Application of glyphosate (N-[phosphonomethyl] glycine) to exporting leaves of sugar beet (Beta vulgaris, L.) during the day lowered stomatal conductance and carbon fixation. Allocation of newly fixed carbon to foliar starch accumulation was nearly completely inhibited, being decreased by the same amount as net carbon fixation. In contrast, decreasing net carbon fixation in untreated leaves by lowering CO₂ concentration caused starch accumulation to decrease, but only in the same proportion as net carbon fixation. Shikimate level increased 50-fold in treated leaves but the elevated rate of carbon accumulation in shikimate was only 4% of the decrease in the rate of starch accumulation. Application of steady state labeling with ¹⁴CO₂ to exporting leaves confirmed the above changes in carbon metabolism, but revealed no other major daytime differences in the ¹⁴C-content of amino acids or other compounds between treated and control leaves. Less ¹⁴C accumulated in treated leaves because of decreased fixation, not increased export. The proportion of newly fixed carbon allocated to sucrose increased, maintaining export at the level in control leaves. Returning net carbon exchange to the rate before treatment restored starch accumulation fully and prevented a decrease in export during the subsequent dark period.     

Effects of decreased net carbon exchange on carbohydrate metabolism in sugar beet source leaves

The relationship between CO₂ concentration and starch synthesis and degradation was studied by measuring leaf starch content and disappearance of ¹⁴C-starch. At a concentration of 340 microliters CO₂ per liter, starch accumulated without degradation of previously synthesized starch. Degradation of starch began when CO₂ concentration was lowered, but its synthesis continued. At 120 microliters CO₂ per liter rates of synthesis and degradation were equal. Even at the CO₂ compensation point, synthesis of starch continued. Concomitant starch synthesis and mobilization supported export from the leaf. Changes in starch metabolism that occur when photosynthesis is CO₂-limited provide a means to study regulation of starch metabolism and carbon allocation in translocating leaves.     

Measurement of Bremsstrahlung radiation for in vivo monitoring of 14C tracer distribution between fruit and roots of kiwifruit (Actinidia arguta) cuttings

In vivo measurements of ¹⁴C tracer distribution have usually involved monitoring the β^? particles produced as ¹⁴C decays. These particles are only detectable over short distances, limiting the use of this technique to thin plant material. In the present experiments, X-ray detectors were used to monitor the Bremsstrahlung radiation emitted since β^? particles were absorbed in plant tissues. Bremsstrahlung radiation is detectable through larger tissue depths. The aim of these experiments was to demonstrate the Bremsstrahlung method by monitoring in vivo tracer-labelled photosynthate partitioning in small kiwifruit (Actinidia arguta (Siebold &; Zucc.) Planch. ex Miq.) plants in response to root pruning. A source shoot, consisting of four leaves, was pulse labelled with ¹⁴CO₂. Detectors monitored import into a fruit and the root system, and export from a source leaf. Repeat pulse labelling enabled the comparison of pre- and post-treatment observations within an individual plant. Diurnal trends were observed in the distribution of tracer, with leaf export reduced at night. Tracer accumulated in the roots declined after approximately 48 h, which may have resulted from export of ¹⁴C from the roots in carbon skeletons. Cutting off half the roots did not affect tracer distribution to the remaining half. Tracer distribution to the fruit was increased after root pruning, demonstrating the higher competitive strength of the fruit than the roots for carbohydrate supply. Increased partitioning to the fruit following root pruning has also been demonstrated in kiwifruit field trials.     

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.     

Sources of sucrose translocated from illuminated sugar beet source leaves

A search for source leaf sucrose pools that differed in their relation to export was carried out in photosynthesizing leaves of Beta vulgaris L. The time course of depletion of [¹⁴C]sucrose in a leaf in unlabeled CO₂ following steady state labeling provided evidence for two distinct sucrose pools. After the start of the light period, leaf blade sucrose remained constant although it exchanged between the two pools. Newly synthesized sucrose destined for export passed through one pool more rapidly than through the other. All of the leaf blade sucrose appeared to exchange with export sucrose. Modeling and regression analysis of [¹⁴C]sucrose data provided a means for estimating the size of the two pools. From 20 to 40% of the sucrose was calculated to be present in the pool that provided the less direct path to export; this was likely vacuolar sucrose. The remainder of the sucrose in the blade was probably in the cytoplasm and veins. Added amounts of leaf blade sucrose, produced in response to elevated CO₂, appeared to be stored mainly in the vacuolar compartment.     

Evidence for Mitochondrial Regulation of Photosynthesis by a Starchless Mutant of Nicotiana sylvestris

Mutant NS458 of Nicotiana sylvestris (Speg. et Comes) contains a defective plastid phosphoglucomutase and accumulates only trace amounts of starch. Determinations of carbon partitioning using tracer d-[3-¹⁴C]glyceric acid showed that the maximal CO₂ assimilation by mature leaves of the mutant at saturating [CO₂] and light and low [O₂] was close to the flux for sucrose formation in the wild type. The mutant is characterized by exceptionally slow oscillations in maximal CO₂ assimilation. The postulate that these slow oscillations follow changes in the cytosolic rate of sucrose phosphate synthesis has been investigated. Studies with wild-type and mutant leaf discs subjected to various treatments failed to indicate that any significant activation-inactivation cycle in sucrose-P synthase activity can occur. The rate of sucrose phosphate synthesis, however, might be altered by variations in the supply of uridine UDP-glucose which is controlled by the rate of ATP regeneration (via UTP regeneration). Treating mutant leaf protoplasts and young leaves with oligomycin, an inhibitor of mitochondrial ATP regeneration, reduced photosynthesis by as much as 25 and 40%, respectively. The wild type failed to show inhibition by oligomycin, i.e. its effect is masked when starch and sucrose synthesis can interact. It is concluded that maximal CO₂ assimilation in the mutant is fine tuned by mitochondrial metabolism such that interactions between sucrose synthesis and mitochondrial processes may generate the observed oscillations.     

Reversal of the Direction of Photosynthate Allocation in Urtica dioica L. Plants by Increasing Cytokinin Import into the Shoot

The natural cytokinin import from the root into the shoot of Urtica dioica plants was enhanced by supplying zeatin riboside (ZR) solutions of various concentrations to a portion less than 10 % of the root system after removal of their tips. After 6 h ZR pretreatment of the plants, ¹⁴CO₂ was supplied for 3 h to a mature (source) leaf or to an expanding leaf and the ¹⁴C-distribution in the whole plant was determined after a subsequent dark period of 14 h. ZR substantially increased ¹⁴C fixation by the expanding leaves and also enhanced export of carbon and transport to the shoot apex. The effect of the hormone treatment was, however, more pronounced when the ¹⁴CO₂ was supplied to a mature leaf. In the control plants these leaves exported carbon only to the roots: When the amount of the natural daily ZR input from the roots to the shoot was enhanced by 20%, the bulk of the ¹⁴C exported from a mature leaf moved to the shoot apex and only a minor portion of ¹⁴C was still detected in the root fraction. A several-fold increase of the natural daily ZR input into the shoot resulted in a flow of ¹⁴C only to the growing parts of the shoot. The results suggest control of the sink strength of the shoot apex by ZR in Urtica diocia.     

Biosynthesis of Sucrose and Mannitol as a Function of Leaf Age in Celery (Apium graveolens L.)

In celery (Apium graveolens L.), the two major translocated carbohydrates are sucrose and the acyclic polyol mannitol. Their metabolism, however, is different and their specific functions are uncertain. To compare their roles in carbon partitioning and sink-source transitions, developmental changes in ¹⁴CO₂ labeling, pool sizes, and key enzyme activities in leaf tissues were examined. The proportion of label in mannitol increased dramatically with leaf maturation whereas that in sucrose remained fairly constant. Mannitol content, however, was high in all leaves and sucrose content increased as leaves developed. Activities of mannose-6-P reductase, cytoplasmic and chloroplastic fructose-1,6-bisphosphatases, sucrose phosphate synthase, and sucrose synthase increased with leaf maturation and decreased as leaves senesced. Ribulose bisphosphate carboxylase and nonreversible glyceraldehyde-3-P dehydrogenase activities rose as leaves developed but did not decrease. Thus, sucrose is produced in all photosynthetically active leaves whereas mannitol is synthesized primarily in mature leaves and stored in all leaves. Onset of sucrose export in celery may result from sucrose accumulation in expanding leaves, but mannitol export is clearly unrelated to mannitol concentration. Mannitol export, however, appears to coincide with increased mannitol biosynthesis. Although mannitol and sucrose arise from a common precursor in celery, subsequent metabolism and transport must be regulated separately.     

The Dynamics of Carbon Supply from Leaves of Barley Plants Grown in Long or Short Days

The role of the mature leaf in supplying carbon for growth inother parts of the plant was examined using a steady-rate ¹⁴CO₂labelling technique. The pattern of events occurring in theleaf during one complete 24 h cycle was compared in plants grownin, and adapted to long and short photoperiods. The rates ofleaf photosynthesis, night respiration and daytime loss of carbonfrom the growing regions of the plant Were similar in long orshort photoperiods. As a percentage of the total carbon fixedduring the photoperiod, total respiration was c. 50% for shortday plants but only 25% for long day plants. Thirty to forty per cent of the carbon fixed during the photoperiodwas retained in the leaf for export during darkness—therest was exported immediately. In leaves of short day plantssucrose and starch were the main form of the stored carbon.By the end of the dark period these compounds had been almostcompletely depleted. In leaves of long day plants there weremuch larger basal levels of sucrose and starch, upon which thediurnal variations were superimposed. These leaves also accumulatedfructosans. The delay in starch remobilization previously foundin leaves of short day plants was also evident in leaves oflong day plants even though large concentrations of sucroseand fructosans were present This suggests the presence of distinctpools of sucrose in the leaf.     

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.     

Up-regulation of sucrose metabolizing enzymes in Oncidium goldiana grown under elevated carbon dioxide

Experiments were conducted in controlled growth chambers to evaluate how increase in CO₂ concentration affected sucrose metabolizing enzymes, especially sucrose phosphate synthase (SPS; EC 2.4.1.14) and sucrose synthase (SS; EC 2.4.1.13), as well as carbon metabolism and partitioning in a tropical epiphytic orchid species (Oncidium goldiana). Response of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) to elevated CO₂ was determined along with dry mass production, photosynthesis rate, chlorophyll content, total nitrogen and total soluble protein content. After 60 days of growth, there was a 80% and 150% increase in dry mass production in plants grown at 750 and 1 100 μl l^?1 CO₂, respectively, compared with those grown at ambient CO₂ (about 370 μl l^?1). A similar increase in photosynthesis rate was detected throughout the growth period when measured under growth CO₂ conditions. Concomitantly, there was a decline in leaf Rubisco activity in plants in elevated CO₂ after 10 days of growth. Over the growth period, leaf SPS and SS activities were up‐regulated by an average of 20% and 40% for plants grown at 750 and 1100 μl l^?1 CO₂, respectively. Leaf sucrose content and starch content were significantly higher throughout the growth period in plants grown at elevated CO₂ than those at ambient CO₂. The partitioning of photosynthetically fixed carbon between sucrose and starch appeared to be unaffected by the 750 μl l^?1 CO₂ treatment, but it was favored into starch under the 1 100 μl l^?1 CO₂ condition. The activities of SPS and SS in leaf extracts were closely associated with photosynthetic rates and with partitioning of carbon between starch and sucrose in leaves. The data are consistent with the hypothesis that the up‐regulation of leaf SPS and SS might be an acclimation response to optimize the utilization and export of organic carbon with the increased rate of inorganic‐carbon fixation in elevated CO₂ conditions.     

Assimilate export by leaves of Ricinus communis L. growing under normal and elevated carbon dioxide concentrations: the same rate during the day, a different rate at night

Castor bean (Ricinus communis L.) plants were grown for 5–7 weeks in a controlled environment at 350 μl l⁻¹ or 700 μl l⁻¹ CO₂. Carbon assimilation, assimilate deposition, dark respiration and assimilate mobilization were measured in leaves 2, 3 and 4 (counted from the base of the plant), and a balance sheet of carbon input and export was elaborated for both CO₂ concentrations. Carbon dioxide assimilation was nearly constant over the illumination period, with only a slight depression occurring at the end of the day in mature source leaves, not in young source leaves. Assimilation was ca. 40% higher at 700 μl l⁻¹ than at 350 μl l⁻¹ CO₂. The source leaves increased steadily in weight per unit area during the first 3 weeks, more at 700 μl l⁻¹ than at 350 μl l⁻¹ CO₂. On top of an irreversible weight increase, there was a large gain in dry weight during the day, which was reversed during the night. This reversible weight gain was constant over the life time of the leaf and ca. 80% higher at 700 μl l⁻¹ than at 350 μl l⁻¹. Most of it was due to carbohydrates. The carbon content (as a percentage) was not altered by the CO₂ treatment. Respiration was 25% higher in high-CO₂ plants when based on leaf area, but the same when based on dry weight. The rate of carbon export via the phloem was the same during the daytime in plants grown at 350 μl l⁻¹ and 700 μl l⁻¹ CO₂. During the night the low-CO₂ plants had only 50% of the daytime export rate, in contrast to the high-CO₂ plants which maintained the high export rate. It was concluded that the phloem loading system is saturated during the daytime in both CO₂ regimes, whereas during the night the assimilate supply is reduced in plants in the normal CO₂ concentration. Two-thirds of the carbon exported from the leaves was permanently incorporated as plant dry matter in the residual plant parts. This “assimilation efficiency” was the same for both CO₂ regimes. It is speculated that under 350 μl l⁻¹ CO₂ the growing Ricinus plant operates at sink limitation during the day and at source limitation during the night. Received: 2 February 1999 / Accepted: 19 April 1999     

Are sucrosyl-oligosaccharides synthesized in mesophyll protoplasts of mature leaves of Cucumis melo?

Biosynthesis of sucrosyl-oligosaccharides (raffinose, stachyose) was traced in source leaves of Cucumis melo after ¹⁴C-photoassimilation. The main carbon compound exported was ¹⁴C-labeled stachyose. No oligosaccharide synthesis was detected in young, importing leaves. Mesophyll protoplasts, isolated from mature leaves which had previously photosynthesized ¹⁴CO₂, did not contain ¹⁴C-oligosaccharides but contained [¹⁴C]-sucrose and ¹⁴C-hexoses. Isolated minor-vein-enriched fractions from the same leaves, however, showed nearly 30% of the ¹⁴C of the neutral fraction to be in oligosaccharides. Isolated, viable mesophyll protoplasts incubated with NaH¹⁴CO₃ also failed to incorporate radioactivity into oligosaccharides, although sucrose and galactinol synthesis was unimpaired. Galactinolsynthase activity in leaf extracts and in mesophyll protoplasts was 16.8 mol·h^-1·mg^-1 protein and 13.8 mol·h^-1·mg^-1 protein, respectively. Galactosyltransferase (EC 2.4.1.67), which synthesizes stachyose from raffinose and galactinol, had an activity of 50 nmol·h^-1·mg^-1 protein in leaf extracts and was also present in the minor-vein-enriched fraction, but could not be detected in mesophyll protoplast lysates. The results indicate that mesophyll cells may not be the site of stachyose synthesis although precursor compounds like sucrose and galactinol are synthesized there.Abbreviation HPLC high-performance liquid chromatography