Thylakoid protein kinase activity and associated control of excitation energy distribution during chloroplast biogenesis in wheat

The activity of thylakoid protein kinase and the regulation of excitation energy distribution between photosystems I and II was examined during chloroplast biogenesis in light-grown Triticum aestivum (wheat) leaves. The specific activity of the thylakoid protein kinase decreased some six-fold during development from the young plastids at the base of the 7-d-old leaf to the mature chloroplasts at the leaf tip. Appreciable activity was also detected in plastids isolated from etiolated leaves. In mature chloroplasts the majority of phosphate was incorporated into the M_r=26,000 apo-proteins of the light-harvesting chlorophyll a/b-protein complex (LHCP). However, at early stages of chloroplast development and in the etioplast, the phosphate was predominantly incorporated into a polypeptide of M_r=9,000 dalton. Immature thylakoids, isolated from the base of the leaf, had relatively low concentrations of LHCP and could perform a State 1-State 2 transition, as demonstrated by ATP-induced quenching of photosystem II fluorescence. Analyses of photosystem I and photosystem II fluorescence-induction curves from intact leaf tissue demonstrated that this transition occurs in vivo at early stages of leaf development and, therefore, may play an important role in regulating energy transduction during chloroplast biogenesis.     

Isolation of intact chloroplasts with high CO2 fixation capacity from sugarbeet leaves containing calcium oxalate

Intact chloroplasts were isolated from sugarbeet leaves by the mechanical disruption technique normally used for spinach. The chloroplast pellet contained a ring of white irregularly shaped crystals which were identified as calcium oxalate. The chloroplasts were greater than 90% intact yet good rates of CO₂ fixation were only obtained when inorganic pyrophosphate or 3-phosphoglycerate were added to the assay medium. Chloroplasts free of calcium oxalate were prepared by purification on a three step Percoll gradient. These purified chloroplasts were highly intact and showed high rates of CO₂ fixation without adding inorganic pyrophosphate or 3-phosphoglycerate. With optimal assay conditions (0.2 mM orthophosphate and pH 8.0) rates of 110–130 μ mole per milligram chlorophyll per hour were routinely obtained. It is concluded that intact chloroplasts capable of high rates of CO₂ fixation can be prepared from sugarbeet leaves using a simple three step Percoll gradient.     

The Distinctive Pattern of Photosystem 2 Activity,Photosynthetic Pigment Accumulation,and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content of Chloroplasts along the Axis of Primary Wheat Leaf Lamina

Wheat (Triticum aestivum L. cv. Sonalika) seedlings were grown in Hoagland solution. Primary leaves were harvested at 8, 12, and 15 d and cut into five equal segments. Contents of photosynthetic pigments and proteins, and photosystem 2 (PS2) activity increased from base to apex of these leaves. Chlorophyll (Chl) content was maximum at 12 d in all the leaf segments, but PS2 activity showed a gradual decline from 8 to 15 d in all leaf segments. In sharp contrast, the CO₂ fixation ability of chloroplasts increased from 8 to 15 d. CO₂ fixation ability of chloroplasts started to decline from base to apex of 15-d-old seedlings, where the content of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBPCO-LSU) increased acropetally. RuBPCO-LSU content was maximum in all the leaf segments in 12-d-old seedlings. This shows a distinctive pattern of PS2, Chl, CO₂ fixation ability of chloroplasts, and RuBPCO-LSU content along the axis of leaf lamina during development and senescence. RuBPCO-LSU (54 kDa) degraded to fragments of 45, 42, 37, 19, and 16 kDa products which accumulated along the leaf axis during ageing of chloroplasts. Thus the CO₂ fixation ability of chloroplasts declines earlier than PS2 activity and photosynthetic pigment contents along the leaf lamina.     

Photosynthetic activity,chloroplast ultrastructure,and leaf characteristics of high-light and low-light plants and of sun and shade leaves

The photosynthetic CO₂-fixation rates, chlorophyll content, chloroplast ultrastructure and other leaf characteristics (e.g. variable fluorescence, stomata density, soluble carbohydrate content) were studied in a comparative way in sun and shade leaves of beech (Fagus sylvatica) and in high-light and low-light seedlings.

1. Sun leaves of the beech possess a smaller leaf area, higher dry weight, lower water content, higher stomata density, higher chlorophyll a/b ratios and are thicker than the shade leaves. Sun leaves on the average contain more chlorophyll in a leaf area unit; the shade leaf exhibits more chlorophyll on a dry weight basis. Sun leaves show higher rates for dark respiration and a higher light saturation of photosynthetic CO₂-fixation. Above 2000 lux they are more efficient in photosynthetic quantum conversion than the shade leaves.
2. The development of HL-radish plants proceeds much faster than that of LL-plants. The cotyledons of HL-plants show a higher dry weight, lower water content, a higher ratio of chlorophyll a/b and a higher gross photosynthesis rate than the cotyledons of the LL-plants, which possess a higher chlorophyll content per dry weight basis. The large area of the HL-cotyledon on the one hand, as well as the higher stomata density and the higher respiration rate in the LL-cotyledon on the other hand, are not in agreement with the characteristics of sun and shade leaves respectively.
3. The development, growth and wilting of wheat leaves and the appearance of the following leaves (leaf succession) is much faster at high quanta fluence rates than in weak light. The chlorophyll content is higher in the HL-leaf per unit leaf area and in the LL-leaf per g dry weight. There are no differences in the stomata density and leaf area between the HL- and LL-leaf. There are fewer differences between HL- and LL-leaves than in beech or radish leaves.
4. The chloroplast ultrastructure of shade-type chloroplasts (shade leaves, LL-leaves) is not only characterized by a much higher number of thylakoids per granum and a higher stacking degree of thylakoids, but also by broader grana than in sun-type chloroplasts (sun leaves, HL-leaves). The chloroplasts of sun leaves and of HL-leaves exhibit large starch grains.
5. Shade leaves and LL-leaves exhibit a higher maximum chlorophyll fluorescence and it takes more time for the fluorescence to decline to the steady state than in sun and HL-leaves. The variable fluorescence VF (ratio of fluorescence decrease to steady state fluorescence) is always higher in the sun and HL-leaf of the same physiological stage (maximum chlorophyll content of the leaf) than in the shade and LL-leaf. The fluorescence emission spectra of sun and HL-leaves show a higher proportion of chlorophyli fluorescence in the second emission maximum F₂ than shade and LL-leaves.
6. The level of soluble carbohydrates (reducing sugars) is significantly higher in sun and HL-leaves than in shade and LL-leaves and even reflects changes in the amounts of the daily incident light.
7. Some but not all characteristics of mature sun and shade leaves are found in HL- and LL-leaves of seedlings. Leaf thickness, dry weight, chlorophyll content, soluble carbohydrate level, photosynthetic CO₂-fixation, height and width of grana stacks and starch content, are good parameters to describe the differences between LL- and HL-leaves; with some reservations concerning age and physiological stage of leaf, a/b ratios, chlorophyll content per leaf area unit and the variable fluorescence are also suitable.

     

Isolation of intact chloroplasts with high CO2 fixation capacity from sugarbeet leaves containing calcium oxalate

Intact chloroplasts were isolated from sugarbeet leaves by the mechanical disruption technique normally used for spinach. The chloroplast pellet contained a ring of white irregularly shaped crystals which were identified as calcium oxalate. The chloroplasts were greater than 90% intact yet good rates of CO₂ fixation were only obtained when inorganic pyrophosphate or 3-phosphoglycerate were added to the assay medium. Chloroplasts free of calcium oxalate were prepared by purification on a three step Percoll gradient. These purified chloroplasts were highly intact and showed high rates of CO₂ fixation without adding inorganic pyrophosphate or 3-phosphoglycerate. With optimal assay conditions (0.2 mM orthophosphate and pH 8.0) rates of 110–130 mole per milligram chlorophyll per hour were routinely obtained. It is concluded that intact chloroplasts capable of high rates of CO₂ fixation can be prepared from sugarbeet leaves using a simple three step Percoll gradient.Abbreviations BSA bovine serum albumin - Chl chlorophyll - Pi inorganic orthophosphate - PPi inorganic pyrophosphate - PGA 3-phosphoglycerate - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(aminoethyl ether) - N,N tetraacetic acid     

Functional and Structural Changes in Senescing Populus deltoides (Bartr.) Chloroplasts

Chloroplasts isolated from Populus deltoides leaves were used to study age-dependent changes in the rate of cyclic photophosphorylation. Single leaves were used to measure CO₂ fixation by leaf discs, chlorophyll concentration, and ATP synthesis. The ability of chloroplasts to synthesize ATP diminished steadily from the time of full leaf expansion, regardless whether the results are expressed on a leaf area or chlorophyll basis. This decline in the rates of ATP synthesis was paralleled by the decline in the rate of CO₂ fixation. The results suggest that the efficiency of the membrane-bound ATP synthesizing system declines with age.     

Plastidic Isoprenoid Synthesis during Chloroplast Development : Change from Metabolic Autonomy to a Division-of-Labor Stage

The chloroplast isoprenoid synthesis of very young leaves is supplied by the plastidic CO₂ → pyruvate → acetyl-coenzyme A (C₃ → C₂) metabolism (D Schulze-Siebert, G Schultz [1987] Plant Physiol 84: 1233-1237) and occurs via the plastidic mevalonate pathway. The plastidic C₃ → C₂ metabolism and/or plastidic mevalonate pathway of barley (Hordeum vulgare L.) seedlings changes from maximal activity at the leaf base (containing developing chloroplasts with incomplete thylakoid stacking but a considerable rate of photosynthetic CO₂-fixation) almost to ineffectivity at the leaf tip (containing mature chloroplasts with maximal photosynthetic activity). The ability to import isopentenyl diphosphate from the extraplastidic space gradually increases to substitute for the loss of endogenous intermediate supply for chloroplast isoprenoid synthesis (change from autonomic to division-of-labor stage). Fatty acid synthesis from NaH¹⁴CO₃ decreases in the same manner as shown for leaf sections and chloroplasts isolated from these. Evidence has been obtained for a drastic decrease of pyruvate decarboxylase-dehydrogenase activity during chloroplast development compared with other anabolic chloroplast pathways (synthesis of aromatic amino acid and branched chain amino acids). The noncompetition of pyruvate and acetate in isotopic dilution studies indicates that both a pyruvate-derived and an acetate-derived compound are simultaneously needed to form introductory intermediates of the mevalonate pathway, presumably acetoacetyl-coenzyme A.     

Photophosphorylation and Carbon Dioxide Fixation by Chloroplasts Isolated from Populus deltoides

A system has been developed for the isolation of photosynthetically active chloroplasts from leaves of Populus deltoides. A high proportion of the chloroplasts appeared intact. The maximum rates of different photosynthetic processes were as follows: CO₂ fixation 3.5 micromoles per milligram chlorophyll per hour, noncyclic ATP synthesis 10 micromoles per milligram chlorophyll per hour, and cyclic ATP synthesis 300 micromoles per milligram chlorophyll per hour.     

Effects of glyoxylate on photosynthesis by intact chloroplasts

Because glyoxylate inhibits CO₂ fixation by intact chloroplasts and purified ribulose bisphosphate carboxylase/oxygenase, glyoxylate might be expected to exert some regulatory effect on photosynthesis. However, ribulose bisphosphate carboxylase activity and activation in intact chloroplasts from Spinacia oleracea L. leaves were not substantially inhibited by 10 millimolar glyoxylate. In the light, the ribulose bisphosphate pool decreased to half when 10 millimolar glyoxylate was present, whereas this pool doubled in the control. When 10 millimolar glyoxylate or formate was present during photosynthesis, the fructose bisphosphate pool in the chloroplasts doubled. Thus, glyoxylate appeared to inhibit the regeneration of ribulose bisphosphate, but not its utilization.

The fixation of CO₂ by intact chloroplasts was inhibited by salts of several weak acids, and the inhibition was more severe at pH 6.0 than at pH 8.0. At pH 6.0, glyoxylate inhibited CO₂ fixation by 50% at 50 micromolar, and glycolate caused 50% inhibition at 150 micromolar. This inhibition of CO₂ fixation seems to be a general effect of salts of weak acids.

Radioactive glyoxylate was reduced to glycolate by chloroplasts more rapidly in the light than in the dark. Glyoxylate reductase (NADP⁺) from intact chloroplast preparations had an apparent K_m (glyoxylate) of 140 micromolar and a V_max of 3 micromoles per minute per milligram chlorophyll.

     

Changes of Anatomical Features, Photosynthesis and Ribulose Bisphosphate Carboxylase-Oxygenase Content of Mango Leaves

Changes in anatomical and physiological features, includingchanges in amount per unit area of anthocyanin and chlorophyll,in leaves of seedling mango (Mangifera indica L. cv. Irwin)trees were determined to understand what controls the rate ofphotosynthesis (Pn) at various stages of development. The youngleaves of seedling trees contained high concentrations of anthocyanin.During enlargement of leaves, the disappearance of anthocyaninand the accumulation of chlorophyll occurred concomitantly;the anthocyanin content began to decrease markedly once theleaf area had reached a maximum. During the early period ofleaf development, the thickness of mesophyll tissue decreasedtemporarily, but when the length of the leaf reached half thatof a mature leaf, the mesophyll began to thicken again. Smallstarch grains appeared in the chloroplasts of the young leavesand chloroplast nucleoids (ct-nuclei) were distributed throughoutthe chloroplasts. When leaves matured, ct-nuclei were displacedto the periphery of chloroplasts because of the accumulationof large starch grains. Compared with young leaves, green andmature leaves contained greater concentrations of ribulose bisphosphatecarboxylase-oxygenase (RuBisCO) protein. The results of immunocytochemicalexamination of RuBisCO under the light microscope reflectedthe results of electrophoresis measurements of RuBisCO. Pn waslow during the chocolate-coloured stage of early leaf development.In green and mature leaves Pn was higher; the average Pn was7·6 mg CO₂ dm^-2 h^-1 under light at intensities above500 µmol m^-2 s^-1.Copyright 1995, 1999 Academic Press Mangifera indica L., mango leaf, chloroplast nucleoids, chloroplast ultrastructure, starch accumulation, anthocyanin, chlorophyll, DAPI staining, SDS-PAGE, immunocytochemical technique     

Photosynthesis and Other Traits in Relation to Chloroplast Number during Soybean Leaf Senescence

Soon after attaining full expansion, soybean (Glycine max [L.] Merr.) leaves enter a senescence phase marked by decline in photosynthetic rate and the progressive loss of chloroplast activity and composition. Our primary goal was to determine if this loss could be accounted for by sequential degradation of whole chloroplasts or by simultaneous degeneration of all chloroplasts. Total photosynthesis (TPs) measured as ¹⁴CO₂ uptake, chloroplast number, ribulose 1,5-bisphosphate carboxylase activity, uncoupled photosynthetic electron transport activity, soluble protein content, and chlorophyll content declined progressively during the 37 days after full leaf expansion. During this period, chloroplast number per unit leaf area was constant for all genotypes studied. We conclude that leaf senescence may be a two-stage process wherein the first stage chloroplast activity and composition declines, but chloroplast numbers do not change. During a brief terminal stage (11 days in our experiment), whole chloroplasts may be lost as well. As a second objective we wished to determine if variation in single-leaf total photosynthetic rate among soybean cultivars is related to corresponding variation in chloroplast number and/or chloroplast activity/composition. By comparing the means for three cultivars known to have rapid leaf TPs and for the three known to have slow TPs, we found the former group to be superior to the latter for all the previously mentioned leaf physiological traits. This superiority was related primarily to differences in chloroplast number and only secondarily to differences in activity and composition per chloroplast.     

Changes in the Number and Composition of Chloroplasts during Senescence of Mesophyll Cells of Attached and Detached Primary Leaves of Wheat (Triticum aestivum L.)

Changes in the number and composition of chloroplasts of mesophyll cells were followed during senescence of the primary leaf of wheat (Triticum aestivum L.). Senescence was due to the natural pattern of leaf ontogeny or was either induced by leaf detachment and incubation in darkness, or incubation of attached leaves in the dark. In each case discrete sections (1 centimeter) of the leaf, representing mesophyll cells of the basal, middle, and tip regions, were examined. For all treatments, senescence was characterized by a loss of chlorophyll and the protein ribulose 1,5-bisphosphate carboxylase (RuBPCase). Chloroplast number per mesophyll cell remained essentially constant during senescence. It was not until more than 80% of the plastid chlorophyll and RuBPCase was degraded that some reduction (22%) in chloroplast number per mesophyll cell was recorded and this was invariably in the mesophyll cells of the leaf tip. We conclude that these data are consistent with the idea that degradation occurs within the chloroplast and that all chloroplasts in a mesophyll cell senesce with a high degree of synchrony rather than each chloroplast senescing sequentially.     

The Rate of Photosynthesis in Isolated Chloroplasts

Chloroplasts were isolated using aqueous and nonaqueous procedures.Aqueous chloroplasts lost approximately 50 per cent, of theirsoluble proteins during isolation. Nonaqueous chloroplasts retainedall their soluble enzymes, but lost their ability to performthe light reactions of photosynthesis. It was possible to reconstitutea chloroplast system of higher activity by adding soluble enzymesfrom nonaqueous chloroplasts to protein-deficient aqueous chloroplasts.The properties of the reconstituted chloroplast system wereas follows: 1. The CO₂ fixation rate of the reconstituted chloroplast system( 4 µM./. chlorophyll/hr.) was 3–4 times that ofthe aqueous chloroplasts ( I µM./. chlorophyll/hr.). Thefixation of aqueous chloroplasts isapparently limited in partby lack of soluble enzymes. 2. During light-fixation, the reconstituted chloroplast systemaccumulated PGA. This indicates that the reduction of PGA totriosephosphate is a rate-limiting step in this system. 3. It was possible to increase the CO₂ fixation to 12 µM.CO₂/mg. chlorophyll/ hr. by addition of ATP and TPNH to thesystem, but the reduction of PGA was still rate-limiting. 4. Further increase in the fixation rate was obtained by concentratingthe reaction mixture. Part of the striking differences of theCO₂-fixing capabilities of chloroplasts in vivo and in vitrois caused by dilution effects. Extrapolation of the dilutioneffect to the protein concentration which exists in chloroplastsyields a CO₂ fixation rate of approximately 30 µM./mg.chlorophyll/hr. 5. Inhibitors which are located in vivo outside the chloroplastsaffect the CO₂ fixation in vitro. 6. Under consideration of the examined factors which influencethe CO₂ fixation of isolated chloroplasts, it is possible toraise the fixation from approximately 1 per cent, to at least15 per cent, of the fixation in vivo.     

Effects of calcium on the photosynthesis of intact leaves and isolated chloroplasts of sugar beets

Effects of calcium on photosynthesis in sugar beets (Beta vulgaris L. cv. F58-554H1) were studied by inducing calcium deficiency and determining changes in CO₂ uptake by attached leaves, electron transport, and photophosphorylation by isolated chloroplasts, and CO₂ assimilation by ribulose diphosphate carboxylase extracts. Calcium deficiency had no significant effect on leaf CO₂ uptake, photoreduction of ferricyanide, cyclic or noncyclic ATP formation of isolated chloroplasts, or on ribulose diphosphate carboxylase CO₂ assimilation, when the rates were expressed per unit chlorophyll. When expressed per unit leaf area CO₂ uptake increased by about 15% in low calcium leaves. The most noticeable effect of calcium deficiency was reduction in leaf area: low calcium had no effect on dark respiratory CO₂ evolution, on leaf diffusion resistance, or on mesophyll resistance to CO₂. We concluded that only small amounts of calcium are required for normal photosynthetic activity of sugar beet leaves.     

Nonphotosynthetic retardation of chloroplast senescence by light

Excised apical portions of green wheat leaf sections were treated with aminotriazole to prevent formation of new chloroplasts. Illumination retarded the decline in chlorophyll content per leaf section, the disintegration of chloroplast ultrastructure, and the loss of capacity for photosynthetic carbon fixation. We interpret these 3 effects of illumination as facets of a single light effect in retarding chloroplast senescence. This light effect in retarding chloroplast senescence has features differing from characteristics of photosynthetic carbon fixation. For example, A) application of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1, 1-dimethylurea did not decrease, and may have even slightly increased, the effectiveness of light; B) although the action spectrum contains peaks in the blue and red regions, it differs from the action spectrum for photosynthetic CO₂ assimilation in wheat; C) in nonphotosynthesizing tissue, application of sugars did not retard chloroplast senescence; D) light saturation was achieved by only a few hundred microwatts/cm². Considered together with the well-known light requirement for chloroplast formation, our results indicate that light has a dual, photomorphogenetic control in maintaining the green status of the plant by also exerting a second effect: retarding of senescence of chloroplasts already present.     

Photosynthesis of Lipids from CO(2) in Spinacia oleracea

Young expanding spinach leaves exposed to ¹⁴CO₂ under physiological conditions for up to 20 minutes assimilated CO₂ into lipids at a mean rate of 7.6 micromoles per milligram chlorophyll per hour following a lag period of 5 minutes. Label entered into all parts of the lipid molecule and only 28% of the ¹⁴C fixed into lipids was found in the fatty acid moieties, i.e. fatty acids were synthesized from CO₂in vivo at a mean rate of 2.1 micromoles per milligram chlorophyll per hour. Intact spinach chloroplasts isolated from these leaves incorporated H¹⁴CO₃ into fatty acids at a maximal rate of 0.6 micromole per milligram chlorophyll per hour, but were unable to synthesize either the polar moieties of their lipids or polyunsaturated fatty acids. Since isolated chloroplasts will only synthesize fatty acids at rates similar to the one obtained with intact leaves in vivo if acetate is used as a precursor, it is suggested that acetate derived from leaf mitochondria is the physiological fatty acid precursor.     

Isolation of Bundle Sheath Cell Chloroplasts from the NADP-ME Type C(4) Plant Zea mays: Capacities for CO(2) Assimilation and Malate Decarboxylation

Bundle sheath chloroplasts have been isolated from Zea mays leaves by a procedure involving enzymic digestion of mechanically prepared strands of bundle sheath cells followed by gentle breakage and filtration. The resulting crude chloroplast preparation was enriched by Percoll density layer centrifugation to yield intact chloroplasts (about 20 micrograms chlorophyll per 10-gram leaf tissue) with high metabolic activities. Based on activities of marker enzymes in the chloroplast and bundle sheath cell extracts, the chloroplasts were essentially free of contamination by other organelles and cytoplasmic material, and were generally about 70% intact. Chlorophyll a/b ratios were high (about 10). With appropriate substrates these chloroplasts displayed high rates of malate decarboxylation, measured as pyruvate formation, and CO₂ assimilation (maximum rates approximately 5 and 3 micromoles per minute per milligram chlorophyll, respectively). These activities were light dependent, linear for at least 20 minutes at 30°C, and displayed highest rates at pH 8.0. High metabolic rates were dependent on addition of an exogenous source of carbon to the photosynthetic carbon reduction cycle (3-phosphoglycerate or dihydroxyacetone phosphate) and a nucleotide (ATP, ADP, or AMP), as well as aspartate. Generally, neither malate decarboxylation nor CO₂ assimilation occurred substantially in the absence of the other activity indicating a close relationship between these processes. Presumably, NADPH required for the photosynthetic carbon reduction cycle is largely supplied during the decarboxylation of malate by NADP-malic enzyme. The results are discussed in relation to the role of bundle sheath chloroplasts in C₄ photosynthesis by species of the NADP-malic enzyme type.     

Photosynthetic CO(2) Fixation at Air Levels of CO(2) by Isolated Spinach Chloroplasts

A system has been developed for the study of photosynthetic CO₂ fixation by isolated spinach chloroplasts at air levels of CO₂. Rates of CO₂ fixation were typically 20 to 60 micromoles/milligrams chlorophyll per hour. The rate of fixation was linear for 10 minutes but then declined to less than 10% of the initial value by 40 minutes. Ribulose 1,5-bisphosphate (RuBP) levels remained unchanged during this period, indicating that they were not the cause for the decline. The initial activity of the RuBP carboxylase in the chloroplast was high for 8 to 10 minutes and then declined similar to the rate of CO₂ fixation, suggesting that the decline in CO₂ fixation may have been caused by deactivation of the enzyme.     

Fixation patterns of 14C within developing leaves of eastern cottonwood

Summary Individual leaves of eastern cottonwood (Populus deltoides), representing an ontogenetic series from leaf plastochron index 0.0 to 8.0, were fed ¹⁴CO₂ photosynthetically and then harvested at times ranging from 15 to 1440 min. The lamina of each fed leaf was sectioned from tip to base into 5 parts, and each part was quantitatively assayed for ¹⁴C activity. In young leaves, the percentage of the total ¹⁴C fixed (expressed in dpm/mg of dry leaf tissue) was high in the lamina tip and decreased almost linearly toward the base. With increasing leaf age, the percentage of ¹⁴C fixed decreased in the lamina tip and increased in the base. The relative activity in mature leaves was almost uniform throughout the lamina. No differences were detected in the ¹⁴C distribution patterns within leaves over the time series.On the basis of the data presented and of anatomical observations of developing cottonwood leaves, the hypothesis that the precociously mature lamina tip may provide photosynthates to the still-expanding lamina base was shown to be invalid. It is concluded that bidirectional transport in a developing cottonwood leaf results from simultaneous import to the immature basal region and export from the mature tip.     

Leaf miner activity and its effects on leaf chemistry in adult beech under elevated CO2

Using the Swiss Canopy Crane CO₂ enrichment facility, the beech weevil's host choice in a CO₂-enriched atmosphere and the in situ effect of its infestation on the chemistry of host leaves were investigated. The fraction of infested leaves was larger on CO₂-enriched trees, suggesting that weevil impact on beech may increase as atmospheric CO₂ concentration keeps rising. Egg insertion in the central leaf vein partially interrupts phloem and xylem transport. Because of this separation between the source of photosynthates, the leaf tip, and the rest of the plant, the leaf tip becomes enriched in non-structural carbohydrates and depleted in nitrogen. Elevated CO₂ slightly enhanced non-structural carbohydrates in uninfested leaves, but exerted no further increase in infested leaf part, suggesting a saturation effect.