Carbon allocation and partitioning in Vigna radiata (L.) Wilczek as affected by additional carbon gain

Carbon allocation to the source leaf, export and partitioning to the sink were studied in mungbean supplied by additional carbon from the source leaves subjected to high CO2 concentrations (600 and 900 cm3 m-3) in three metabolic and functional source-sink combinations. The plants were pruned to a source-path-sink system. With CO2 enrichment there was an appreciable increase in net photosynthetic CO2 uptake in earlier formed and physiologically younger leaves. Most of the carbon fixed as a result of enrichment was translocated out of the source leaf within one diurnal cycle. The carbon remaining in the source leaf was unchanged. Partitioning of extra carbon into starch or sugar depended upon the amount of extra carbon synthesized. The unloading of the extra carbon into sinks depended on whether it was used for growth or stored. Under increased carbon content, the leaf as a sink was able to reorganize its metabolic reactions more rapidly to maintain the required gradient for unloading than the pod acting as the sink.     

The fate of apoplastic sucrose in sink and source leaves of Urtica dioica

Experiments were performed with developing and mature leaves of Urtica dioica L. to trace differences which could be interpreted in terms of cell wall-bound acid invertase (EC 3.2.1.26) participating in phloem unloading in a sink leaf. The pH of apoplastic fluid that was collected by gentle centrifugation of entire leaves was identical (7.1) in the two types of leaves; also, fluorometric determination with esculetin showed a neutral apoplastic pH between 7.0 in the source and 7.2 in the sink leaf. To detect whether differences in apoplastic pH occur within limited leaf areas, such as of the tissue surrounding the veins, the metabolic fate of [¹⁴C]–(fructosyl)-sucrose that was administered via the xylem was investigated. In source leaves, there was a large transitory decrease in [¹⁴C]-sucrose followed by a substantial resynthesis of this compound. In sink leaves, resynthesis was less significant and carbon was incorporated mainly in starch, charged soluble compounds and cell walls. However, after correction for resynthesis, the two types of leaves showed an identical capacity for sucrose cleavage. Finally, activation of the apoplastic invertase by administering labelled sucrose in buffered solution of pH 5.0 did not result in an enhanced degradation. By contrast, apoplastic fluid collected from leaves which had been infiltrated with buffer solutions of pH 5.5 and 8.0, respectively, showed a rapid adjustment of the pH close to the natural neutral value by the mesophyll tissue. The results are incompatible with the idea of an active invertase in the sink (and the source) leaves apoplast, and hence do not lend support to the theory of apoplastic cleavage of sucrose being required for phloem unloading in this kind of a utilization sink.     

Sucrose-phosphate synthase responds differently to source-sink relations and to photosynthetic rates: Lolium perenne L. growing at elevated pCO2 in the field

Lolium perenne, a main component species in managed grassland, is well adapted to defoliation, fertilization, and regrowth cycles; and hence, to changes in the assimilatory carbon source‐sink ratio. In the Swiss Free Air CO₂ Enrichment experiment the source‐sink ratio is (i) increased by elevated partial pressure of CO₂ (p_CO2), (ii) decreased by enhanced carbon use under high N fertilization, and (iii) gradually increased during regrowth after defoliation. Since sucrose synthesis plays a central role in leaf carbohydrate metabolism in this fructan‐accumulating species, we investigated how sucrose‐phosphate synthase (SPS) responds to the differing assimilatory carbon fluxes and source‐sink ratios in the field. Assimilatory carbon flux, as estimated by leaf gas exchange, strongly depended on p_CO2. Surprisingly, the SPS content per leaf area did not increase with p_CO2, but increased with N fertilization. During later regrowth, when a dense canopy had formed, the SPS content decreased; in particular, SPS was decreased at high N under elevated p_CO2. Further, the higher assimilatory carbon flux through SPS at elevated p_CO2 was accompanied by a higher activation state of SPS. The SPS content correlated very strongly with the ratio of free sucrose to free amino acid in leaves, which represents the carbon source‐sink ratio. Hence, SPS content in L. perenne appears to be regulated by the current, strongly nitrogen‐dependent, source‐sink relation.     

Phloem Unloading in Sink Leaves of Nicotiana benthamiana: Comparison of a Fluorescent Solute with a Fluorescent Virus

Using noninvasive imaging techniques, we compared phloem unloading of the membrane-impermeant, fluorescent solute carboxyfluorescein (CF) with that of potato virus X expressing the gene for the green fluorescent protein. Although systemic virus transport took considerably longer to occur than did CF transport, unloading of both solute and virus occurred predominantly from the class III vein network, a highly branched veinal system found between class II veins. The minor veins (classes IV and V) played no role in solute or virus import but were shown to be functional in xylem transport at the time of import by labeling with Texas Red dextran. After virus exit from the class III phloem, the minor veins eventually became infected by cell-to-cell virus movement from the mesophyll. During the sink/source transition, phloem unloading of CF was inhibited from class III veins before the cessation of phloem import through them, suggesting a symplastic isolation of the phloem in class III veins before its involvement in export. The progression of the sink/source transition for carbon was unaffected by the presence of the virus in the sink leaf. However, the virus was unable to cross the sink/source boundary for carbon that was present at the time of viral entry, suggesting a limited capacity for cell-to-cell virus movement into the apical (source) region of the leaf. A functional model of the sink/source transition in Nicotiana benthamiana is presented. This model provides a framework for the analysis of solute and virus movement in leaves.     

Phloem unloading in tobacco sink leaves: insensitivity to anoxia indicates a symplastic pathway

Phloem unloading in transition sink leaves of tobacco (Nicotiana tabacum L.) was analyzed by quantitative autoradiography. Detectable levels of labeled photoassimilates entered sink leaves approx. 1 h after source leaves were provided with ¹⁴CO₂. Samples of tissue were removed from sink leaves when label was first detected and further samples were taken at the end of an experimental phloem-unloading period. The amount of label in veins and in surrounding cells was determined by microdensitometry of autoradiographs using a microspectrophotometer. Photoassimilate unloaded from first-, second-and third-order veins but not from smaller veins. Import termination in individual veins was gradual. Import by the sink leaf was completely inhibited by exposing the sink leaf to anaerobic conditions, by placing the entire plant in the cold, or by steam-girdling the sink-leaf petiole. Phloem unloading was completely inhibited by cold; however, phloem unloading continued when the sink-leaf petiole was steam girdled or when the sink leaf was exposed to a N₂ atmosphere. Compartmental efflux-analysis indicated that only a small percentage of labeled nutrients was present in the free space after unloading from sink-leaf veins in a N₂ atmosphere. The results are consistent with passive symplastic transfer of photoassimilates from phloem to surrounding cells.Symbol VI radio of ¹⁴C in veins and interveinal tissue     

Sink strength regulates photosynthesis in sugarcane

The relationship in sugarcane (Saccharum spp.) between photosynthetic source tissue and sink material was examined through manipulation of the sink:source ratio of field-grown Saccharum spp. hybrid cv. N19 (N19). To enhance sink strength, all leaves, except for the third fully expanded leaf, were enclosed in 90% shade cloth for varying periods of time. Variations in sucrose, glucose and fructose concentrations were measured and the effects of shading on the leaf gas exchange and fluorescence characteristics recorded. Changes in carbon partitioning caused by shading were examined based on the uptake and translocation of fixed 14CO2. Following a decline in sucrose concentrations in young internodal tissue and shaded leaves, significant increases in the CO2-saturated photosynthetic rate (Jmax), carboxylation efficiency (CE) and electron transport rate were observed in unshaded leaves after 8 d of shading treatment. It was concluded that up-regulation of source-leaf photosynthetic capacity is correlated with a decrease in assimilate availability to acropetal culm sink tissue. Furthermore, a significant relationship was revealed between source hexose concentration and photosynthetic activity.     

Partitioning of 14C-labeled photosynthate to allelochemicals and primary metabolites in source and sink leaves of aspen: evidence for secondary metabolite turnover

Theories on allelochemical concentrations in plants are often based upon the relative carbon costs and benefits of multiple metabolic fractions. Tests of these theories often rely on measuring metabolite concentrations, but frequently overlook priorities in carbon partitioning. We conducted a pulse-labeling experiment to follow the partitioning of ¹⁴CO₂-labeled photosynthate into ten metabolic pools representing growth and maintenance (amino acids, organic acids, lipids plus pigments, protein, residue), defense (phenolic glycosides, methanol:water and acetone-soluble tannins/phenolics), and transport and storage (sugars and starch) in source and importing sink leaves of quaking aspen (Populus tremuloides). The peak period of ¹⁴C incorporation into sink leaves occurred at 24 h. Within 48 h of labeling, the specific radioactivity (dpm/mg dry leaf weight) of phenolic glycosides declined by over one-third in source and sink leaves. In addition, the specific radioactivity in the tannin/phenolic fraction decreased by 53% and 28% in source and sink leaves, respectively. On a percent recovery basis, sink leaves partitioned 1.7 times as much labeled photosynthate into phenolic glycosides as source leaves at peak ¹⁴C incorporation. In contrast, source leaves partitioned 1.8 times as much ¹⁴C-labeled photosynthate into tannins/phenolics as importing sink leaves. At the end of the 7-day chase period, sink leaves retained 18%, 52%, and 30% of imported ¹⁴C photosynthate, and labeled source leaves retained 15%, 66%, and 19% of in situ photosynthate in metabolic fractions representing transport and storage, growth and maintenance, and defense, respectively. Analyses of the phenolic fractions showed that total phenolics were twice as great and condensed tannins were 1.7 times greater in sink than in source leaves. The concentration of total phenolics and condensed tannins did not change in source and sink leaves during the 7-day chase period. Received: 31 July 1998 / Accepted: 8 February 1999     

Phloem Unloading in Developing Leaves of Sugar Beet : II. Termination of Phloem Unloading

Phloem unloading in developing leaves of Beta vulgaris L. (`Klein E' multigerm) occurred from successively higher order branches of veins as leaves matured. Phloem unloading was studied in autoradiographs of leaf samples taken at various times during the arrival of a pulse of ¹⁴C-labeled photoassimilate. Extension of mass flow of sieve element contents into leaf vein branches was determined from the high level of radiolabel in veins soon after first arrival of the pulse. Rapid entry, indicative of mass flow through open sieve pores, occurred down to the fourth division of veins in young, importing leaves and to the fifth or terminal branch in importing regions near the zone of transition from sink to source. The rate of unloading decreased with leaf age, as evidenced by the increased time required for the vein-mesophyll demarcation to become obscured. The rate of import per unit leaf area, measured by steady state labeling with ¹⁴CO₂ also decreased as a leaf matured. The decline in import appeared to result from progressive changes that increased resistance to unloading of sieve elements and eventually terminated phloem unloading.     

Effects of carbohydrate accumulation on photosynthesis differ between sink and source leaves of Phaseolus vulgaris L

Accumulation of non-structural carbohydrate in leaves represses photosynthesis. However, the extent of repression should be different between sink leaves (sugar consumers) and source leaves (sugar exporters). We investigated the effects of carbohydrate accumulation on photosynthesis in the primary leaves of bean (Phaseolus vulgaris L.) during leaf expansion. To increase the carbohydrate content of the leaves, we supplied 20 mM sucrose solution to the roots for 5 d (sugar treatment). Plants supplied only with water and nutrients were used as controls. The carbohydrate contents, which are the sum of glucose, sucrose and starch, of the sugar-treated leaves were 1.5-3 times of those of the control leaves at all developmental stages. In the young sink leaves, the photosynthetic rate at saturating light and at an ambient CO2 concentration (A360) did not differ between the sugar-treated and control leaves. The A360 of sugar-treated source leaves gradually decreased relative to the control source leaves with leaf expansion. The initial slope of the A-Ci (CO2 concentration in the intercellular space) curve, and the Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) content per leaf area showed trends similar to that of A360. Differences in Amax between the treatments were slightly smaller than those in A360. These results indicate that the effect of carbohydrate accumulation on photosynthesis is significant in the source leaves, but not in the young sink leaves, and that the decrease in Rubisco content was the main cause of the carbohydrate repression of photosynthesis.     

Reproductive effort and floral photosynthesis in Spiranthes cernua (Orchidaceae)

This study examined the cost of reproduction and photosynthetic characteristics of the reproductive structures of Spiranthes cernua, an agamospermic, terrestrial orchid. Reproduction was frequent: two-thirds of the plants flowered at least 2 yr in a row and one-fourth of the consecutive-year runs were ~3 yr. Neither a significant decrease in leaf area nor a reduced likelihood of flowering was observed following 1 or 2 yr of inflorescence production. While there was a tendency for plants producing >16 flowers to have decreased size the next year, plants with the greatest number of flowers (31+) were the most likely to reproduce. Leaf and reproductive gas exchange were measured in the field. Low but positive rates of net photosynthesis were documented at all stages of inflorescence development. The average rates of photosynthesis for each stage were: leaves, 9.2 mmol CO2/m2s; inflorescence in bud, 3.7 mmol CO2/m2s; inflorescence in flower, 2.5 mmol CO2/m2s and infructescence, 0.2 mmol CO2/m2s. Based on diurnal gas exchange, the contribution of leaves and reproductive structures to seasonal carbon assimilation was 91.6 and 8.4%, respectively. The role of the inflorescence as a source and sink for carbon assimilation may lower the cost of reproduction and support frequent inflorescence production.     

Long Distance Translocation of Sucrose, Serine, Leucine, Lysine, and Carbon Dioxide Assimilates: II. Oats

To establish whether several amino acids were equally able to enter the phloem of oat (Avena sativa L.) plants and be transported, several (14)C-labeled amino acids were applied individually to an abraded spot on a fully expanded source leaf. The base of an immature sink leaf was monitored with a GM tube for time and rate of arrival of radioactivity. Transport of (14)C-sucrose and (14)CO(2) assimilates was measured for a comparison. The applied l-serine, l-lysine, and l-leucine, as well as sucrose, entered the phloem and were transported to the sink leaf at rates between 1.16 and 1.83 cm/min. Transport velocity for CO(2) assimilates was 1.57 cm/min. A heat girdle near the top of the source leaf sheath blocked most transport, which indicated that transport was primarily through the phloem. Mass transfer rates for amino acids were only 3% as great as that for sucrose, suggesting different mechanisms of entry for sucrose than for amino acids into the phloem. The higher percentage of CO(2) assimilates mobilized to the sink leaf was attributed to the greater surface area of minor veins accessible to loading, as compared to those compounds supplied via an abraded spot. Serine was extensively metabolized in the source leaf, and radioactive products in the sink leaf mirrored those in the source leaf. Most radioactivity of lysine and leucine remained within these compounds in the source, path, and sink tissues. We concluded that there was no barrier to entry of amino acids into the phloem and transport therein. Data do not suggest a specific mechanism for entry of amino acids into the phloem.     

Phloem Unloading in Developing Leaves of Sugar Beet : I. Evidence for Pathway through the Symplast

Physiological and transport data are presented in support of a symplastic pathway of phloem unloading in importing leaves of Beta vulgaris L. (`Klein E multigerm'). The sulfhydryl reagent p-chloromercuribenzene sulfonic acid (PCMBS) at concentration of 10 millimolar inhibited uptake of exogenous [¹⁴C]sucrose by sink leaf tissue over sucrose concentrations of 0.1 to 5.0 millimolar. Inhibited uptake was 24% of controls. The same PCMBS treatment did not affect import of ¹⁴C-label into sink leaves during steady state labeling of a source leaf with ¹⁴CO₂. Lack of inhibition of import implies that sucrose did not pass through the free space during unloading. A passively transported xenobiotic sugar, l-[¹⁴C]glucose, imported by a sink leaf through the phloem, was evenly distributed throughout the leaf as seen by whole-leaf autoradiography. In contrast, l-[¹⁴C]glucose supplied to the apoplast through the cut petiole or into a vein of a sink leaf collected mainly in the vicinity of the major veins with little entering the mesophyll. These patterns are best explained by transport through the symplast from phloem to mesophyll.     

Cytokinin deficiency causes distinct changes of sink and source parameters in tobacco shoots and roots

Cytokinin deficiency causes pleiotropic developmental changes such as reduced shoot and increased root growth. It was investigated whether cytokinin-deficient tobacco plants, which overproduce different cytokinin oxidase/dehydrogenase enzymes, show changes in different sink and source parameters, which could be causally related to the establishment of the cytokinin deficiency syndrome. Ultrastructural analysis revealed distinct changes in differentiating shoot tissues, including an increased vacuolation and an earlier differentiation of plastids, which showed partially disorganized thylakoid structures later in development. A comparison of the ploidy levels revealed an increased population of cells with a 4C DNA content during early stages of leaf development, indicating an inhibited progression from G2 to mitosis. To compare physiological characteristics of sink leaves, source leaves and roots of wild-type and cytokinin-deficient plants, several photosynthetic parameters, content of soluble sugars, starch and adenylates, as well as activities of enzymes of carbon assimilation and dissimilation were determined. Leaves of cytokinin-deficient plants contained less chlorophyll and non-photochemical quenching of young leaves was increased. However, absorption rate, photosynthetic capacity (F(v)/F(m) and J(CO2 max)) and efficiency (Phi CO(2 app)), as well as the content of soluble sugars, were not strongly altered in source leaves, indicating that chlorophyll is not limiting for photoassimilation and suggesting that source strength did not restrict shoot growth. By contrast, shoot sink tissues showed drastically reduced contents of soluble sugars, decreased activities of vacuolar invertases, and a reduced ATP content. These results strongly support a function of cytokinin in regulating shoot sink strength and its reduction may be a cause of the altered shoot phenotype. Roots of cytokinin-deficient plants contained less sugar compared with wild-type. However, this did not negatively affect glycolysis, ATP content, or root development. It is suggested that cytokinin-mediated regulation of the sink strength differs between roots and shoots.     

Carbon partitioning in Arabidopsis thaliana is a dynamic process controlled by the plants metabolic status and its circadian clock

Plant growth involves the coordinated distribution of carbon resources both towards structural components and towards storage compounds that assure a steady carbon supply over the complete diurnal cycle. We used ¹⁴CO₂ labelling to track assimilated carbon in both source and sink tissues. Source tissues exhibit large variations in carbon allocation throughout the light period. The most prominent change was detected in partitioning towards starch, being low in the morning and more than double later in the day. Export into sink tissues showed reciprocal changes. Fewer and smaller changes in carbon allocation occurred in sink tissues where, in most respects, carbon was partitioned similarly, whether the sink leaf assimilated it through photosynthesis or imported it from source leaves. Mutants deficient in the production or remobilization of leaf starch exhibited major alterations in carbon allocation. Low‐starch mutants that suffer from carbon starvation at night allocated much more carbon into neutral sugars and had higher rates of export than the wild type, partly because of the reduced allocation into starch, but also because of reduced allocation into structural components. Moreover, mutants deficient in the plant's circadian system showed considerable changes in their carbon partitioning pattern suggesting control by the circadian clock.     

Physiology of maize I: A comprehensive and integrated view of nitrogen metabolism in a C4 plant

Maize ( Zea mays L., line F2) plants were grown in the field under high or low fertilization input to monitor the metabolic, biochemical and molecular events occurring in young vegetative leaves and in the different leaf stages along the main axis in plants harvested 15 days after silking. This study shows that in maize which possess large sinks represented by the seeds, nitrogen (N) management is different compared with tobacco in which sink strength is much lower and mostly limited to young developing leaves. Although in young leaves nitrate assimilation predominates in both species, ammonium assimilation exhibits some species-specific differences with respect to inorganic and organic N metabolite accumulation during leaf ageing. These differences are likely to be related to the high sink strength of the ear in maize, which continuously imports carbon and N assimilates during grain filling. Consequently, a number of cytosolic glutamine synthetase isoenzymes are expressed during leaf ageing to maintain a constant flux of reduced N necessary for the synthesis of organic N molecules used either for leaf protein synthesis or directly translocated to the grain. This situation contrasts with that found in tobacco for which leaf ammonium assimilation in the plastids is shifted to the cytosol during the transition from sink leaves to source leaves. These species-specific differences for N assimilation and recycling are discussed in relation to the evolution of leaf photosynthetic activity and leaf senescence, which both seem to be largely dependent on the different sink strength in each species.     

Sugar Selectivity and Other Characteristics of Phloem Loading in Beta vulgaris L

The rate of phloem loading, its selectivity, and the disposition of labeled carbon were studied following application of (14)C-labeled sugars to the free space of source leaves of sugar beet (Beta vulgaris L.). Buffered 10 mm solutions of (14)C-labeled sucrose, fructose, stachyose, mannitol, 3-0-methyl glucose or l-glucose were applied to the abraded epidermis of source leaves held in the dark. Distribution of the labeled carbon from sugar taken up from the free space was studied by micro-densitometry of autoradiographs. Uptake of labeled sugar from the free space, partition between mesophyll and minor veins, metabolic conversions, export and respiration were followed during the 3-hr time course studies. Rates of sugar uptake into the minor veins, flux rates through the sieve element-companion cell complex membrane and concentration ratios between free space and the interior of the minor vein phloem cells were compared for the six sugars studied for evidence of active uptake. The composition of the free space solution in leaves photosynthesizing in (14)CO(2) was studied by vacuum infiltration of the source leaf air spaces and removal of the solution by centrifugation. Labeled compounds in this solution were compared to those in an aqueous ethanol extract of the same leaf pieces.The results in sugar beet source leaves support the concept of direct, active uptake of sucrose from free space into minor veins. This is not the case for fructose, 3-0-methyl glucose, mannitol, or stachyose. The latter two sugars, which are translocated in some plants, are not loaded into the minor veins at a rate sufficient to make them a significant component of the material translocated. The rate of phloem loading is controlled in part by mesophyll metabolism, especially as it affects the availability of sucrose to the free space. Both the rate and selectivity of export are controlled by uptake from the free space into the sieve element-companion cell complex of the minor veins.     

L-Ascorbic acid is accumulated in source leaf phloem and transported to sink tissues in plants

L-Ascorbic acid (AsA) was found to be loaded into phloem of source leaves and transported to sink tissues. When L-[(14)C]AsA was applied to leaves of intact plants of three different species, autoradiographs and HPLC analysis demonstrated that AsA was accumulated into phloem and transported to root tips, shoots, and floral organs, but not to mature leaves. AsA was also directly detected in Arabidopsis sieve tube sap collected from an English green aphid (Sitobion avenae) stylet. Feeding a single leaf of intact Arabidopsis or Medicago sativa with 10 or 20 mM L-galactono-1,4-lactone (GAL-L), the immediate precursor of AsA, lead to a 7- to 8-fold increase in AsA in the treated leaf and a 2- to 3-fold increase of AsA in untreated sink tissues of the same plant. The amount of AsA produced in treated leaves and accumulated in sink tissues was proportional to the amount of GAL-L applied. Studies of the ability of organs to produce AsA from GAL-L showed mature leaves have a 3- to 10-fold higher biosynthetic capacity and much lower AsA turnover rate than sink tissues. The results indicate AsA transporters reside in the phloem, and that AsA translocation is likely required to meet AsA demands of rapidly growing non-photosynthetic tissues. This study also demonstrates that source leaf AsA biosynthesis is limited by substrate availability rather than biosynthetic capacity, and sink AsA levels may be limited to some extent by source production. Phloem translocation of AsA may be one factor regulating sink development because AsA is critical to cell division/growth.