Translocation and Metabolic Conversion of C-Labeled Assimilates in Detached and Attached Leaves of Phaseolus vulgaris L. in Different Phases of Leaf Expansion

Leaf excision greatly affected the actual levels of ¹⁴C-assimilates in laminas and petioles of primary bean leaves (Phaseolus vulgaris L.) following a transport period. However, it did not affect the percentage of starch in the insoluble residue; starch decreased from 20% of the insoluble residue after exposure to ¹⁴CO₂ to 3% after 5 hr in both attached and detached leaves. The transition from import to export of attached and detached leaves was at the same stage, i.e., when the cotyledons were 63 to 85% depleted. The composition of the ¹⁴C-assimilates in importing leaves was different from that in exporting leaves. In the former, only 5% of the soluble label was free sugar, while 74% was free sugar in the latter. The failure of importing leaves to export was not due to the labeled substances being nontransferable. Extracts from importing leaves applied to exporting leaves were exported; these extracts were high in amino acids and organic acids but low in free sugar. However, exporting leaves exposed to ¹⁴CO₂ appeared to export sugars more readily than amino acids. Cotyledon excision did not delay transition of leaves from import to export. Actually, excision seemed to enhance slightly the transition of the primary leaves from import to export.     

Diurnal changes in allocation of newly fixed carbon in exporting sugar beet leaves

Storage of newly fixed carbon as starch and sucrose follows a regular daily pattern in exporting sugar beet leaves under constant day length and level of illumination. Up to the final two hours of the light period, when starch storage declines, a nearly constant proportion of newly fixed carbon was allocated to carbohydrate storage, principally starch. Sucrose is stored only early in the light period, when there is little accumulation of starch. Pulse labeling with ¹⁴CO₂ revealed that considerable starch synthesis was taking place at this time. Starch made the previous day was not mobilized during this period but breakdown of newly synthesized starch may occur when carbon flow into sucrose synthesis increases early in the day. At the end of the day, starch storage declined from the constant level observed during most of the day, but no diversion of label into export of specific alternative compounds could be detected. Lowered storage of starch persisted when the 14-hour light period was lengthened. Changed allocation of recently fixed carbon to sucrose and starch at the beginning and end of the light period was not the result of outright inactivation of pathways but of regulation of carbon flow.     

A Method for Calculating Sucrose Synthesis Rates throughout a Light Period in Sugar Beet Leaves

Sucrose synthesis rate in an exporting sugar beet (Beta vulgaris L.) leaf was calculated from simultaneous measurements of export and changes in leaf sucrose level. The amount of recently fixed carbon exported was determined from net carbon assimilated minus the tracer carbon accumulated in the leaf. The relative amount of ¹⁴C accumulated in the leaf supplied with ¹⁴CO₂ throughout an entire light period was recorded continuously with a Geiger-Mueller detector. To produce a continuous time course for tracer carbon accumulated in the leaf during the light period, the latter curve was superimposed on values for tracer carbon accumulated in leaves sampled at hourly intervals. Validity of the method requires that nearly all of the carbon that is exported be sucrose and that nearly all of the sucrose that is synthesized be either exported or accumulated as sucrose in the exporting leaves. These conditions appeared to be fulfilled in the situations where the method was applied. The method was used to study the effect of increasing atmospheric CO₂ concentration on the rate of sucrose synthesis. Further, the method can be used in conjunction with the gathering of other data such as gas exchange, metabolite levels, and enzyme activities in a set of leaves of a similar age on the same plant. This assemblage of data was found to be useful for understanding how rates of photosynthesis, sucrose synthesis, and translocation are regulated in relation to each other in an intact plant.     

Photosynthate supply and utilization in alfalfa : a developmental shift from a source to a sink limitation of photosynthesis

Long-term carbon dioxide enrichment, ¹⁴CO₂ feeding, and partial defoliation were employed as probes to investigate source/sink limitations of photosynthesis during the development of symbiotically grown alfalfa. In the mature crop, long-term CO₂ enrichment does not affect the rates of net photosynthesis, relative growth, ¹⁴C export to nonphotosynthetic organs, or the rates of ¹⁴C label incorporation into leaf sucrose, starch, or malate. The rate of glycolate labeling is, however, substantially reduced under these conditions. When the mature crop was partially defoliated, a considerable increase in net photosynthesis occurred in the remaining leaves. In the seedling crop, long-term CO₂ enrichment increased dry matter accumulation, primarily as a result of increases in leaf starch content. Although the higher rates of starch synthesis are not maintained, the growth enhancement of the enriched plants persisted throughout the experimental period. These results imply a source limitation of seedling photosynthesis and a sink limitation of photosynthesis in more mature plants. Consequently, both the supply and the utilization of photosynthate may limit seasonal photosynthesis in alfalfa.     

Diurnal starch accumulation and utilization in phosphorus-deficient soybean plants

The effects of phosphorus deficiency on carbohydrate accumulation and utilization in 34-day-old soybean (Glycine max L. Merr.) plants were characterized over a diurnal cycle to evaluate the mechanisms by which phosphorus deficiency restricts plant growth. Phosphorus deficiency decreased the net CO₂ exchange rate throughout the light period. The decrease in the CO₂ exhange rate was associated with a decrease in stomatal conductance and an increase in the internal CO₂ concentration. These observations indicate that phosphorus deficiency increased mesophyll resistance. Assimilate export rate from the youngest fully expanded leaves was decreased by phosphorus deficiency, whereas starch concentrations in these leaves were increased. Higher starch concentrations in phosphorus-deficient youngest fully expanded leaves resulted from a longer period of net starch accumulation and a shorter period of net starch degradation relative to those for phosphorus-sufficient controls. Phosphorus deficiency decreased sucrose-P synthase activity by 27% (averaged over the diurnal cycle), and essentially eliminated diurnal variation in sucrose-P-synthase activity. Diurnal variations in nonstructural carbohydrate concentrations in leaves and stems were also less pronounced in phosphorus-deficient plants than in controls. In phosphorus-deficient plants, only 30% of the whole plant starch present at the end of a light phase was utilized during the subsequent 12-hour dark phase as compared with 68% for phosphorus-sufficient controls. Although phosphorus deficiency decreased the CO₂ exchange rate and whole plant leaf area, accumulation of high starch concentrations in leaves and stems and restricted starch utilization in the dark indicate that growth processes (i.e. sink activities) were restricted to a greater extent than photosynthetic capacity. Further experimentation is required to determine whether decreased starch utilization in phosphorus-deficient plants is the cause or the result of restricted growth.     

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.     

Photosynthesis and Carbon Partitioning in Transgenic Tobacco Plants Deficient in Leaf Cytosolic Pyruvate Kinase

Whole-plant diurnal C exchange analysis provided a noninvasive estimation of daily net C gain in transgenic tobacco (Nicotiana tabacum L.) plants deficient in leaf cytosolic pyruvate kinase (PK_c−). PK_c− plants cultivated under a low light intensity (100 μmol m⁻² s⁻¹) were previously shown to exhibit markedly reduced root growth, as well as delayed shoot and flower development when compared with plants having wild-type levels of PK_c (PK_c+). PK_c− and PK_c+ source leaves showed a similar net C gain, photosynthesis over a range of light intensities, and a capacity to export newly fixed ¹⁴CO₂ during photosynthesis. However, during growth under low light the nighttime, export of previously fixed ¹⁴CO₂ by fully expanded PK_c− leaves was 40% lower, whereas concurrent respiratory ¹⁴CO₂ evolution was 40% higher than that of PK_c+ leaves. This provides a rationale for the reduced root growth of the PK_c− plants grown at low irradiance. Leaf photosynthetic and export characteristics in PK_c− and PK_c+ plants raised in a greenhouse during winter months resembled those of plants grown in chambers at low irradiance. The data suggest that PK_c in source leaves has a critical role in regulating nighttime respiration particularly when the available pool of photoassimilates for export and leaf respiratory processes are low.     

Diurnal changes in allocation and partitioning of recently assimilated carbon in loblolly pine seedlings

We examined diurnal fluctuations in acquisition and partitioning of recently assimilated ¹⁴CO₂, and in subsequent allocation and partitioning to roots of loblolly pine (Pinus taeda L.) seedlings. Nonmycorrhizal seedlings were grown under optimal nutrient conditions in continuously flowin solution culture. Shoots of 15-week-old loblolly pine seedlings were labeled with ¹⁴CO₂ for 30 min at four separate labeling times: 1000, 1200, 1400 and 1600 h. Six whole plant harvests were conducted during a 48 h chase period, i.e. 0, 4, 8 12, 24 and 48 h after the end of the labeling and evacuation periods. Although assimilation of ¹⁴CO₂ was constant between 1000 and 1400 h, there were significant differences in partitioning of ¹⁴C-labeled assimilate in needles of all age classes. The highest percentage of recently assimilated ¹⁴CO₂ in the ethanol-soluble fraction of photosynthesizing tissue was observed near the beginning and end of the photoperiod. Partitioning of ¹⁴C in the ethanol-soluble fraction declined between the 1000 and 1400 h labeling eriods, and was accompanied by an increase in partitioning of recently assimilated ¹⁴CO₂ toward starch and a decrease in respiratory losses. These data suggest that most of the ¹⁴CO₂ assimilated at 1000 h was used to support shoot metabolic activities and possibly restore soluble sugar reserves. Peak starch accumulation in needles during the 1400 h labeling period, concomitant with minimal respiratory loss, indicated that photosynthate production exceeded demand and export out of source leaves. A possible feedback regulation of photosynthesis by starch and/or sugar accumulation may be responsible for the observed decline in assimilation of ¹⁴CO₂ during the 1600 h labeling period. Net accumulation of recently assimilated ¹⁴CO₂ in roots was correlated with assimilation rate of ¹⁴CO₂, but independent of partitioning of recently assimilated carbon in photosynthetic tissue. However, the percentage of total seedling ¹⁴C allocated to roots was essentially the same throughout the 48 h chase, regardless of time of labeling and assimilation rate. The data suggest a strong diurnal regulation of starch and soluble sugars synthesized from recently assimilated carbon in needles of loblolly pine seedlings that was independent of assimilation rate. Allocation and transport of recently assimilated carbon to roots of loblolly pine seedlings were not subject to short-term fluctuations in supply and demand.     

Relationships between Carbon Assimilation, Partitioning, and Export in Leaves of Two Soybean Cultivars

To evaluate leaf carbon balance during rapid pod-fill in soybean (Glycine max [L.] Merrill), measurements were made of CO₂ assimilation at mid-day and changes in specific leaf weight, starch, and sucrose concentrations over a 9-hour interval. Assimilate export was estimated from CO₂ assimilation and leaf dry matter accumulation. Chamber-grown `Amsoy 71' and `Wells' plants were subjected on the day of the measurements to one of six photosynthetic photon flux densities in order to vary CO₂ assimilation rates.

Rate of accumulation of leaf dry matter and rate of export both increased as CO₂ assimilation rate increased in each cultivar.

Starch concentrations were greater in Amsoy 71 than in Wells at all CO₂ assimilation rates. At low CO₂ assimilation rates, export rates in Amsoy 71 were maintained in excess of 1.0 milligram CH₂O per square decimeter leaf area per hour at the expense of leaf reserves. In Wells, however, export rate continued to decline with decreasing CO₂ assimilation rate. The low leaf starch concentration in Wells at low CO₂ assimilation rates may have limited export by limiting carbon from starch remobilization.

Both cultivars exhibited positive correlations between CO₂ assimilation rate and sucrose concentration, and between sucrose concentration and export rate. Carbon fixation and carbon partitioning both influenced export rate via effects on sucrose concentration.

     

The H+-Sucrose Cotransporter NtSUT1 Is Essential for Sugar Export from Tobacco Leaves

In many species translocation of sucrose from the mesophyll to the phloem is carrier mediated. A sucrose/H⁺-symporter cDNA, NtSUT1, was isolated from tobacco (Nicotiana tabacum) and shown to be highly expressed in mature leaves and at low levels in other tissues, including floral organs. To study the in vivo function of NtSUT1, tobacco plants were transformed with a SUT1 antisense construct under control of the cauliflower mosaic virus 35S promoter. Upon maturation, leaves of transformants expressing reduced amounts of SUT1 mRNA curled downward, and strongly affected plants developed chloroses and necroses that led to death. The leaves exhibited impaired ability to export recently fixed ¹⁴CO₂ and were unable to export transient starch during extended periods of darkness. As a consequence, soluble carbohydrates accumulated and photosynthesis was reduced. Autoradiographs of leaves show a heterogenous pattern of CO₂ fixation even after a 24-h chase. The ¹⁴C pattern does not change with time, suggesting that movement of photosynthate between mesophyll cells may also be impaired. The affected lines show a reduction in the development of the root system and delayed or impaired flowering. Taken together, the effects observed in a seed plant (tobacco) demonstrate the importance of SUT1 for sucrose loading into the phloem via an apoplastic route and possibly for intermesophyll transport as well.     

Comparison of ethylenediaminetetraacetate-enhanced exudation from detached and translocation from attached bean leaves

A technique for collection of phloem exudate from detached leaves using 20 millimolar EDTA (pH 7.0) has previously been developed (King, Zeevaart 1974 Plant Physiol 53: 96-103). It was the aim of the present study to determine the efficiency of this technique in relation to undisturbed export from attached leaves. Paired primary leaves of bean seedlings (Phaseolus vulgaris L. cv Montcalm) were used to minimize variations in plant material. Attached leaves, exposed to ¹⁴CO₂ for 10 minutes with subsequent excision of one of the leaves and collection of the exudate over a 12-hour period, showed a 25% export of total assimilated ¹⁴C from the attached versus 15% of total assimilated ¹⁴C in the form of exudation from the detached ones. Leaf excision changed the labeling pattern within the leaf, increasing% total leaf ¹⁴C-activity in the ethanolic fraction, while decreasing activity in the starch fraction, as compared to attached leaves. This was presumably caused by a lack of translocation from the detached leaves. Excision did not affect dark respiration. However, measurements of total nonstructural carbohydrates in leaf starch and neutral fractions indicated no significant differences between attached and leaves detached in EDTA. Thus, in terms of actual carbon export, and accompanying distribution of nonexported carbohydrate within the leaf, EDTA-enhanced exudation compares favorably with translocation from attached leaves.     

Reactivation of phloem export in mature maize leaves after a dark period

Mature leaves of corn plants (Zea mays L. cv. Prior) which were darkened for 48 h contain neither bundle-sheath starch nor glucose, and their sucrose content is below 5 M. In such leaves phloem export has ceased. When re-illuminated, photosynthetic sucrose production starts without delay, but the sucrose: glucose ratio is 1.25:1. Obviously, most of the new-formed sugar is utilized locally. Labeling with ¹⁴CO₂ has shown that phloen export starts 30 to 40 min after the onset of photosynthesis, when the sucrose: glucose ratio has increased to 13:1. The first newly formed starch can be detected when phloem export is reactivated. Glucose content remains constantly low af about 2 M for at least 2 h, and it never exceeds 10 M. Radioactivity in the exporting veins is about five times higher after 2 to 7 h of re-illumination than in the 14-h-day plant. Therefore, phloem export is either intensified during the period of reactivation or exported assimilates are partly unloaded along their way. Comparison of photosynthetic activity of equal-sized leaf strips has shown that both accumulation of photosynthates and radioactivity of exporting veins are about three times higher in the detached strip than in the strip which remained attached to the mother plant.     

The effect of sulphite on the regulation of photosynthetic sucrose synthesis in poplar leaves

Sulphite at concentrations from 0.05 to 5.0 mM was supplied to illuminated, detached poplar (Populus deltoides Bart. ex Marsh) leaves via the transpiration stream. The rate of CO2 fixation and partitioning of newly fixed carbon between sucrose and starch were measured and compared with the contents of selected phosphorylated intermediates, the contents of fructose-2,6-bisphosphate (Fru2,6BP) and the activation of sucrose-phosphate synthase (SPS). Supplying leaves with 0.5 mM sulphite led to an increase in the sucrose/starch partitioning ratio without altering the rate of ¹⁴CO2 fixation. The increase in sucrose synthesis compared to starch synthesis was accompanied by relatively small changes of 3-phosphoglyceric acid (PGA), fructose-1,6-bisphosphate (Fru1,6BP), hexose phosphates (hexose-)), uridine 5'-diphosphoglucose (UDPGlc), an accumulation of triose phosphates (triose-P), an activation of SPS, and decreased Fru2,6BP contents. Supplying leaves with 1.0 mM sulphite decreased ¹⁴CO2 assimilation and increased partitioning of fixed carbon into starch. A selective inhibition of sucrose synthesis was accompanied by an accumulation of triose-P, Fru1,6BP, hexose-P, and a decrease of PGA contents. There was also a large increase of Fru2,6BP contents and a decline in the activation of SPS. It could be argued that sulphite affects the allocation of photosynthetic carbon to sucrose and that sulphite can inhibit photosynthesis via a selective inhibition of sucrose synthesis.     

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.     

LEAF DEVELOPMENT,PHOTOSYNTHESIS, AND C14 DISTRIBUTION IN POPULUS DELTOIDES SEEDLINGS

Rates of net photosynthesis and dark respiration and distribution of C¹⁴ from selected leaves were determined for young cottonwood (Populus deltoides) trees at different stages of development. Four series of five trees—one series for each of four treated leaf positions—were included in the study. Maximum C¹⁴ export occurred when a leaf had just attained maximum size. Lower stem leaves reached maturity quickly and began exporting photosynthate when demands of the young seedling were high. Leaves at higher stem positions matured more slowly, but senescence was also delayed so their effective export life was prolonged. Translocation from a newly exporting leaf was primarily upward to developing leaves and the apex. As a leaf at any one position aged, the translocation pattern gradually shifted from upward to bi-directional and finally to a predominantly downward direction. Photosynthate translocated downward was incorporated into stem wood and roots. Maximum photosynthetic efficiency coincided with the downward shift of C¹⁴ export. Thereafter, net photosynthesis began to decline, at first slowly and then more rapidly. The patterns of photosynthesis, respiration, and C¹⁴ export associated with leaf age all varied according to leaf position on the stem.     

Overexpression of sucrose-phosphate synthase in tomato plants grown with CO2 enrichment leads to decreased foliar carbohydrate accumulation relative to untransformed controls

Tomato plants expressing the maize sucrose-phosphate synthase (SPS) cDNA under the control of the promoterof the small subunit of ribulose-1,5-bisphosphate carboxylase oxygenase (rbcS) promoter were grown 5 weeks in air (450 μmol.m^–2.s^–1 irradiance, 350 ppm CO₂) and then either maintained in air or exposed to CO₂ enrichment (1 000 ppm CO₂) for 8 d. A linear relationship between the foliar sucrose to starch ratio and maximal extractable SPS activity was found both in air and high CO₂. Starch accumulation was dramatically increased in all plants subjected to CO₂ enrichment but the CO₂-dependent increase in foliar starch accumulation was much lower in the leaves of the SPS transformants than in those of the untransformed controls in the same conditions. Maximal extractable ribulose-1,5-bisphosphate carboxylase/oxygenase activity was reduced by growth at high CO₂ to a similar extent in both plant types. The carbon/nitrogen ratios were similar in both plant lines in both growth conditions after 20 d exposure to high CO₂. A small (5 %) increase in carbon export capacity was observed at high CO₂ in the leaves of transformed plants compared to leaves from untransformed controls. Increased foliar SPS activity did not, however, prevent acclimation of photosynthesis in plants grown with long-term CO₂ enrichment.     

High-light effects on CO2 fixation gradients across leaves

Chlorophyll fluorescence and internal patterns of ¹⁴CO₂ fixation were measured in sun and shade leaves of spinach after treatment with various light intensities. When sun leaves were irradiated with 2000μmol m^?2 s^?1 for 2h, F_V/F_M decreased by about 15%, but ¹⁴CO₂ fixation was unaffected, whereas shade leaves exhibited a 21% decrease in F_v/F_M and a 25% decrease in ¹⁴CO₂ fixation. Irradiation of sun and shade leaves with 4000μmol m^?1 for 4 h decreased F_V/F_M by 30% in sun leaves and 40% in shade leaves, while total ¹⁴CO₂ fixation decreased by 41% in sun leaves and 55% in shade leaves. After light treatment, gradients of CO₂ fixation across leaves were determined by measuring ¹⁴CO₂ fixed in paradermal leaf sections after a 10s pulse of ¹⁴CO₂. Gradients of ¹⁴CO₂ fixation in control sun and shade leaves were identified when expressed on a relative basis and normalized for leaf depth. Treatment of leaves with 2000 μmol PAR m^?2 s^?1 for 2h did not after patterns of carbon fixation across sun leaves, but slightly altered the pattern in shade leaves. In contrast, treatment of sun and shade leaves with 4000μmol m^?2 s^?1 for 4h decreased carbon fixation more in the palisade mesophyll cells than in the spongy mesophyll cells of sun and shade leaves, and fixation in medial tissue of shade leaves was dramatically decreased compared to the adaxial and abaxial tissue. The interaction between leaf anatomy and biochemical parameters involved in tolerance to photoinhibition in spinach is discussed.     

Effect of altered sink: source ratio on photosynthetic metabolism of source leaves

When seven crop species were grown under identical environmental conditions, decreased sink:source ratio led to a decreased photosynthetic rate within 1 to 3 days in Cucumis sativus L., Gossypium hirsutum L., and Raphanus sativus L., but not in Capsicum annuum L., Solanum melongena L., Phaseolus vulgaris L., or Ricinus communis L. The decrease was not associated with stomatal closure. In cotton and cucumber, sink removal led to an increase in starch and sugar content, in glucose 6-phosphate and fructose 6-phosphate pools, and in the proportion of ¹⁴C detected in sugar phosphates and UDPglucose following ¹⁴CO₂ supply. When mannose was supplied to leaf discs to sequester cytoplasmic inorganic phosphate, promotion of starch synthesis, and inhibition of CO₂ fixation, were observed in control discs, but not in discs from treated plants. Phosphate buffer reduced starch synthesis in the latter, but not the former discs. The findings suggest that sink removal led to a decreased ratio inorganic phosphate:phosphorylated compounds. In beans ¹⁴C in sugar phosphates increased following sink removal, but without sucrose accumulation, suggesting tighter feedback control of sugar level. Starch accumulated to higher levels than in the other plants, but CO₂ fixation rate was constant for several days.     

Evidence for circadian regulation of starch and sucrose synthesis in sugar beet leaves

Starch accumulation and sucrose synthesis and export were measured in leaves of sugar beet (Beta vulgaris L.) during a period of prolonged irradiance in which illumination was extended beyond the usual 14-hour day period. During much of the 14-hour day period, approximately 50% of the newly fixed carbon was distributed to sucrose, about 40% to starch, and less than 10% to hexose. Beginning about 2 hours before the end of the usual light period, the portion of newly fixed carbon allocated to sucrose gradually increased, and correspondingly less carbon went to starch. By the time the transition ended, about 4 hours into the extension of the light period, nearly 90% of newly fixed carbon was incorporated into sucrose and little or none into starch. Most of the additional sucrose was exported. Gradual cessation of starch accumulation was not the result of a futile cycle of simultaneous starch synthesis and degradation. Neither was it the result of a decrease in the extractable activity of adenosine diphosphoglucose pyrophosphorylase or phosphoglucose isomerase, enzymes important in starch synthesis. Nor was there a notable change in control metabolites considered to be important in regulating starch synthesis. Starch accumulation appeared to decrease markedly because of an endogenous circadian shift in carbon allocation, which occurred in preparation for the usual night period and which diverted carbon from the chloroplast to the cytosol and sucrose synthesis.