Control of carbon partitioning and photosynthesis by the triose phosphate/phosphate translocator in transgenic tobacco plants (Nicotiana tabacum L.). I. Comparative physiological analysis of tobacco plants with antisense repression and overexpression of the triose phosphate/phosphate translocator

 The physiological properties of transgenic tobacco plants (Nicotiana tabacum L.) with decreased or increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT) were compared in order to investigate the extent to which the TPT controls metabolic fluxes in wild-type tobacco. For this purpose, tobacco lines with an antisense repression of the endogenous TPT (αTPT) and tobacco lines overexpressing the TPT gene isolated from the C₄ plant Flaveria trinervia (FtTPT) were used. The F. trinervia TPT expressed in yeast cells exhibited transport characteristics identical to the TPT from C₃ plants. Neither antisense TPT plants nor FtTPT overexpressors showed a phenotype when grown in a greenhouse in air. Contents of starch and soluble sugars in upper source leaves were similar in TPT underexpressors and FtTPT overexpressors compared to the wild type at the end of the photoperiod. The FtTPT overexpressors incorporated more ¹⁴CO₂ in sucrose than the wild type, indicating that the TPT limits sucrose biosynthesis in the wild type. There were only small effects on labelling of amino acids and organic acids. The mobilisation of starch was enhanced in αTPT lines but decreased in FtTPT overexpressors compared to the wild type. Enzymes involved in starch mobilisation or utilisation, such as α-amylase or hexokinase were increased in αTPT plants and, in the case of amylases, decreased in FtTPT overexpressors. Moreover, α-amylase activity exhibited a pronounced diurnal variation in αTPT lines with a maximum activity after 8 h in the light. These changes in starch hydrolytic activities were confirmed by activity staining of native gels. Activities of glucan phosphorylases were unaffected by either a decrease or an increase in TPT activity. There were also effects of TPT activities on steady-state levels of phosphorylated intermediates as well as total amino acids and malate. In air, there was no or little effect of altered TPT transport activity on either rates of photosynthetic electron transport and/or CO₂ assimilation. However, in elevated CO₂ (1500 μl · l⁻¹) and low O₂ (2%) the rate of CO₂ assimilation was decreased in the αTPT lines and was slightly higher in FtTPT lines. This shows that the TPT limits maximum rates of photosynthesis in the wild type. Received: 26 March 1999 / Accepted: 21 August 1999     

Manipulation of triose phosphate/phosphate translocator and cytosolic fructose-1,6-bisphosphatase,the key components in photosynthetic sucrose synthesis,enhances the source capacity of transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Photoassimilated carbons are converted to sucrose in green plant leaves and distributed to non-phototropic tissues to provide carbon and energy. In photosynthetic sucrose biosynthesis, the chloroplast envelope triose phosphate/phosphate translocator (TPT) and cytosolic fructose-1,6-bisphosphatase (cFBPase) are key components in photosynthetic sucrose biosynthesis. The simultaneous overexpression of TPT and cFBPase was utilized to increase the source capacity of Arabidopsis. The TPT and cFBPase overexpression lines exhibited enhanced growth with larger rosette sizes and increased fresh weights compared with wild-type (WT) plants. The simultaneous overexpression of TPT and cFBPase resulted in enhanced photosynthetic CO₂ assimilation rates in moderate and elevated light conditions. During the phototropic period, the soluble sugar (sucrose, glucose, and fructose) levels in the leaves of these transgenic lines were also higher than those of the WT plants. These results suggest that the simultaneous overexpression of TPT and cFBPase enhances source capacity and consequently leads to growth enhancement in transgenic plants.     

Overexpression of the triose phosphate translocator (TPT) complements the abnormal metabolism and development of plastidial glycolytic glyceraldehyde‐3‐phosphate dehydrogenase mutants

The presence of two glycolytic pathways working in parallel in plastids and cytosol has complicated the understanding of this essential process in plant cells, especially the integration of the plastidial pathway into the metabolism of heterotrophic and autotrophic organs. It is assumed that this integration is achieved by transport systems, which exchange glycolytic intermediates across plastidial membranes. However, it is unknown whether plastidial and cytosolic pools of 3‐phosphoglycerate (3‐PGA) can equilibrate in non‐photosynthetic tissues. To resolve this question, we employed Arabidopsis mutants of the plastidial glycolytic isoforms of glyceraldehyde‐3‐phosphate dehydrogenase (GAPCp) that express the triose phosphate translocator (TPT) under the control of the 35S (35S:TPT) or the native GAPCp1 (GAPCp1:TPT) promoters. TPT expression under the control of both promoters complemented the vegetative developmental defects and metabolic disorders of the GAPCp double mutants (gapcp1gapcp2). However, as the 35S is poorly expressed in the tapetum, full vegetative and reproductive complementation of gapcp1gapcp2 was achieved only by transforming this mutant with the GAPCp1:TPT construct. Our results indicate that the main function of GAPCp is to supply 3‐PGA for anabolic pathways in plastids of heterotrophic cells and suggest that the plastidial glycolysis may contribute to fatty acid biosynthesis in seeds. They also suggest a 3‐PGA deficiency in the plastids of gapcp1gapcp2, and that 3‐PGA pools between cytosol and plastid do not equilibrate in heterotrophic cells.     

Growth, ageing and death of a photoautotrophic plant cell culture

 Batch cultures of photoautotrophic cell suspensions of Chenopodiumrubrum L., growing in an inorganic medium on CO₂ under a daily balanced light–dark regime of 16 : 8 h could be maintained for approximately 100 d without subcultivation. The long-lived cultures showed an initial cell division phase of 4 weeks, followed by a stationary phase of another 4 weeks, after which ageing and progressive cell death reduced the number of living cells and the cultures usually expired after another 3–4 weeks. These developmental phases of the cell culture were characterised with respect to photosynthetic performance, dark respiration, content of phytohormones and capacity of cell division. Cell division of the majority of the cells finished in the G1- or G0-phase of the cell cycle, caused by a pronounced decline in the endogenous levels of auxin and cytokinins. Supply of these growth factors to resting cells resulted in resumption of cytokinesis, at least by some of the cells. However, responsiveness to the phytohomones declined during the stationary phase, and subcultivation was no longer possible beyond day 60 when the phases of ageing and death commenced. Ageing was characterised by a further decline in the photosynthetic capacity of the cells, by a climacteric enhancement of dark respiration, but also by a slight increase in the level of IAA and cytokinins concomitant with a decrease in ethylene. Similarities and differences between the development of batch-cultured photoautotrophic cells of C. rubrum and that of a leaf are discussed with respect to using the cell culture as a model for a leaf. Received: 30 April 1999 / Accepted: 21 August 1999     

Photosynthetic carbon metabolism in leaves of transgenic tobacco (Nicotiana tabacum L.) containing decreased amounts of fructose 2,6-bisphosphate

The aim of this work was to examine the role of fructose 2,6-bisphosphate (Fru-2,6-P₂) in photosynthetic carbon partitioning. The amount of Fru-2,6-P₂ in leaves of tobacco (Nicotiana tabacum L. cv. Samsun) was reduced by introduction of a modified mammalian gene encoding a functional fructose-2,6-bisphosphatase (EC 3.1.3.46). Expression of this gene in transgenic plants reduced the Fru-2,6-P₂ content of darkened leaves to between 54% and 80% of that in untransformed plants. During the first 30 min of photosynthesis sucrose accumulated more rapidly in the transgenic lines than in the untransformed plants, whereas starch production was slower in the transgenic plants. On illumination, the proportion of ¹⁴CO₂ converted to sucrose was greater in leaf disks of transgenic lines possessing reduced amounts of Fru-2,6-P₂ than in those of the control plants, and there was a corresponding decrease in the proportion of carbon assimilated to starch in the transgenic lines. Furthermore, plants with smaller amounts of Fru-2,6-P₂ had lower rates of net CO₂ assimilation. In illuminated leaves, decreasing the amount of Fru-2,6-P₂ resulted in greater amounts of hexose phosphates, but smaller amounts of 3-phosphoglycerate and dihydroxyacetone phosphate. These differences are interpreted in terms of decreased inhibition of cytosolic fructose-1,6-bisphosphatase resulting from the lowered Fru-2,6-P₂ content. The data provide direct evidence for the importance of Fru-2,6-P₂ in co-ordinating chloroplastic and cytosolic carbohydrate metabolism in leaves in the light. Received: 8 February 2000 / Accepted: 25 April 2000     

Photosynthetic acclimation of maize to growth under elevated levels of carbon dioxide

The effects of elevated CO₂ concentrations on the photochemistry, biochemistry and physiology of C₄ photosynthesis were studied in maize (Zea mays L.). Plants were grown at ambient (350 μL L⁻¹) or ca. 3 times ambient (1100 μL L⁻¹) CO₂ levels under high light conditions in a greenhouse for 30 d. Relative to plants grown at ambient CO₂ levels, plants grown under elevated CO₂ accumulated ca. 20% more biomass and 23% more leaf area. When measured at the CO₂ concentration of growth, mature leaves of high-CO₂-grown plants had higher light-saturated rates of photosynthesis (ca. 15%), lower stomatal conductance (71%), higher water-use efficiency (225%) and higher dark respiration rates (100%). High-CO₂-grown plants had lower carboxylation efficiencies (23%), measured under limiting CO₂, and lower leaf protein contents (22%). Activities of a number of C₃ and C₄ cycle enzymes decreased on a leaf-area basis in the high-CO₂-grown plants by 5–30%, with NADP-malate dehydrogenase exhibiting the greatest decrease. In contrast, activities of fructose 1,6-bisphosphatase and ADP-glucose pyrophosphorylase increased significantly under elevated CO₂ condition (8% and 36%, respectively). These data show that the C₄ plant maize may benefit from elevated CO₂ through acclimation in the capacities of certain photosynthetic enzymes. The increased capacity to synthesize sucrose and starch, and to utilize these end-products of photosynthesis to produce extra energy by respiration, may contribute to the enhanced growth of maize under elevated CO₂. Received: 30 April 1999 / Accepted: 17 June 1999     

Decrease of phosphoribulokinase activity by antisense RNA in transgenic tobacco: definition of the light environment under which phosphoribulokinase is not in large excess

 To test the hypothesis that the contribution of phosphoribulokinase (PRK) to the control of photosynthesis changes depending on the light environment of the plant, the response of transgenic tobacco (Nicotiana tabacum L.) transformed with antisense PRK constructs to irradiance was determined. In plants grown under low irradiance (330 μmol m⁻² s⁻¹) steady-state photosynthesis was limited in plants with decreased PRK activity upon exposure to higher irradiance, with a control coefficient of PRK for CO₂ assimilation of 0.25 at and above 800 μmol m⁻² s⁻¹. The flux control coefficient of PRK for steady-state CO₂ assimilation was zero, however, at all irradiances in plant material grown at 800 μmol m⁻² s⁻¹ and in plants grown in a glasshouse during mid-summer (alternating shade and sun 300–1600 μmol m⁻² s⁻¹). To explain these differences between plants grown under low and high irradiances, Calvin cycle enzyme activities and metabolite content were determined. Activities of PRK and other non-equilibrium Calvin cycle enzymes fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase and ribulose-1,5-bisphosphate carboxylase-oxygenase were twofold higher in plants grown at 800 μmol m⁻² s⁻¹ or in the glasshouse than in plants grown at 330 μmol m⁻² s⁻¹. Activities of equilibrium enzymes transketolase, aldolase, ribulose-5-phosphate epimerase and isomerase were very similar under all growth irradiances. The flux control coefficient of 0.25 in plants grown at 330 μmol m⁻² s⁻¹ can be explained because low ribulose-5-phosphate content in combination with low PRK activity limits the synthesis of ribulose-1,5-bisphosphate. This limitation is overcome in high-light-grown plants because of the large relative increase in activities of sedoheptulose-1,7-bisphosphatase and fructose-1,6-bisphosphatase under these conditions, which facilitates the synthesis of larger amounts of ribulose-5-phosphate. This potential limitation will have maintained evolutionary selection pressure for high concentrations of PRK within the chloroplast. Received: 15 November 1999 / Accepted: 27 January 2000     

Interseasonal comparison of CO2 concentrations, isotopic composition, and carbon dynamics in an Amazonian rainforest (French Guiana)

Canopy CO₂ concentrations in a tropical rainforest in French Guiana were measured continuously for 5 days during the 1994 dry season and the 1995 wet season. Carbon dioxide concentrations ([CO₂]) throughout the canopy (0.02–38 m) showed a distinct daily pattern, were well-stratified and decreased with increasing height into the canopy. During both seasons, daytime [CO₂] in the upper and middle canopy decreased on average 7–10 μmol mol⁻¹ below tropospheric baseline values measured at Barbados. Within the main part of the canopy (≥ 0.7 m), [CO₂] did not differ between the wet and dry seasons. In contrast, [CO₂] below 0.7 m were generally higher during the dry season, resulting in larger [CO₂] gradients. Supporting this observation, soil CO₂ efflux was on average higher during the dry season than during the wet season, either due to diffusive limitations and/or to oxygen deficiency of root and microbial respiration. Soil respiration rates decreased by 40% after strong rain events, resulting in a rapid decrease in canopy [CO₂] immediately above the forest floor of about 50␣μmol mol⁻¹. Temporal and spatial variations in [CO₂]_canopy were reflected in changes of δ¹³C_canopy and δ¹⁸O_canopy values. Tight relationships were observed between δ¹³C and δ¹⁸O of canopy CO₂ during both seasons (r ² > 0.86). The most depleted δ¹³C_canopy and δ¹⁸O_canopy values were measured immediately above the forest floor (δ¹³C = −16.4‰; δ¹⁸O = 39.1‰ SMOW). Gradients in the isotope ratios of CO₂ between the top of the canopy and the forest floor ranged between 2.0‰ and 6.3‰ for δ¹³C, and between 1.0‰ and 3.5‰ for δ¹⁸O. The δ¹³C_leaf and calculated c _i/c _a of foliage at three different positions were similar for the dry and wet seasons indicating that the canopy maintained a constant ratio of photosynthesis to stomatal conductance. About 20% of the differences in δ¹³C_leaf within the canopy was accounted for by source air effects, the remaining 80% must be due to changes in c _i/c _a. Plotting 1/[CO₂] vs. the corresponding δ¹³C ratios resulted in very tight, linear relationships (r ² = 0.99), with no significant differences between the two seasons, suggesting negligible seasonal variability in turbulent mixing relative to ecosystem gas exchange. The intercepts of these relationships that should be indicative of the δ¹³C of respired sources were close to the measured δ¹³C of soil respired CO₂ and to the δ¹³C of litter and soil organic matter. Estimates of carbon isotope discrimination of the entire ecosystem, Δ_e, were calculated as 20.3‰ during the dry season and as 20.5‰ during the wet season. Received: 3 March 1996 / Accepted: 19 October 1996     

Flexible coupling between light-dependent electron and vectorial proton transport in illuminated leaves of C3 plants. Role of photosystem I-dependent proton pumping

The role of cyclic electron transport has been re-examined in leaves of C₃ plants because the bioenergetics of chloroplasts (H⁺/e = 3 in the presence of a Q-cycle; H⁺/ATP = 4 of ATP synthesis) had suggested that cyclic electron flow has no function in C₃ photosynthesis. After light activation of pea leaves, the dark reduction of P700 (the donor pigment of PSI) following far-red oxidation was much accelerated. This corresponded to loss of sensitivity of P700 to oxidation by far-red light and a large increase in the number of electrons available to reduce P700⁺ in the dark. At low CO₂ and O₂ molar ratios, far-red light was capable of decreasing the activity of photosystem II (measured as the ratio of variable to maximal chlorophyll fluorescence, F_v/F_m) and of increasing light scattering at 535 nm and zeaxanthin synthesis, indicating formation of a transthylakoid pH gradient. Both the light-induced increase in the number of electrons capable of reducing far-red-oxidised P700 and the decline in F_v/F_m brought about by far-red in leaves were prevented by methyl viologen. Antimycin A inhibited CO₂-dependent O₂ evolution of pea leaves at saturating but not under limiting light; in its presence, far-red light failed to decrease F_v/F_m. The results indicate that cyclic electron flow regulates the quantum yield of photosystem II by decreasing the intrathylakoid pH when there is a reduction in the availability of electron acceptors at the PSI level (e.g. during drought or cold stresses). It also provides ATP for the carbon-reduction cycle under high light. Under these conditions, the Q-cycle is not able to maintain a H⁺/e ratio of 3 for ATP synthesis: we suggest that the ratio is flexible, not obligatory. Received: 23 February 1999 / Accepted: 19 August 1999     

The relative importance of tryptophan-dependent and tryptophan-independent biosynthesis of indole-3-acetic acid in tobacco during vegetative growth

A quantitative study of indole-3-acetic acid (IAA) turnover, and the contribution of tryptophan-dependent and tryptophan-independent IAA-biosynthesis pathways, was carried out using protoplast preparations and shoot apices obtained from wild-type and transgenic, IAA-overproducing tobacco (Nicotiana tabacum L.) plants, during a phase of growth when the level of endogenous IAA was stable. Based on the rate of disappearance of [¹³C₆]IAA, the half-life of the IAA pool was calculated to be 1.1 h in wild-type protoplasts and 0.8 h in protoplasts from the IAA-overproducing line, corresponding to metabolic rates of 59 and 160 pg IAA (μg Chl)⁻¹ h⁻¹, respectively. The rate of conversion of tryptophan to IAA was 15 pg IAA (μg Chl)⁻¹ h⁻¹ in wild-type protoplasts and 101 pg IAA (μg Chl)⁻¹ h⁻¹ in protoplasts from IAA-overproducing plants. In both instances, IAA was metabolised more rapidly than it was synthesised from tryptophan. As the endogenous IAA pools were in a steady state, these findings indicate that IAA biosynthesis via the tryptophan-independent pathway was 44 pg IAA (μg Chl)⁻¹ h⁻¹ and 59 pg IAA (μg Chl)⁻¹ h⁻¹, respectively, in the wild-type and transformed protoplast preparations. In a parallel study with apical shoot tissue, the presumed site of IAA biosynthesis, the rate of tryptophan-dependent IAA biosynthesis exceeded the rate of metabolism of [¹³C₆]IAA despite the steady state of the endogenous IAA pool. The most likely explanation for this anomaly is that, unlike the protoplast system, injection of substrates into the apical tissues did not result in uniform distribution of label, and that at least some of the [²H₅]tryptophan was metabolised in compartments not normally active in IAA biosynthesis. This demonstrates the importance of using experimental systems where labelling of the precursor pool can be strictly controlled. Received: 18 January 2000 / Accepted 24 February 2000     

Stem respiration of ponderosa pines grown in contrasting climates: implications for global climate change

We examined the effects of climate and allocation patterns on stem respiration in ponderosa pine (Pinus ponderosa) growing on identical substrate in the cool, moist Sierra Nevada mountains and the warm, dry, Great Basin Desert. These environments are representative of current climatic conditions and those predicted to accompany a doubling of atmospheric CO₂, respectively, throughout the range of many western north American conifers. A previous study found that trees growing in the desert allocate proportionally more biomass to sapwood and less to leaf area than montane trees. We tested the hypothesis that respiration rates of sapwood are lower in desert trees than in montane trees due to reduced stem maintenance respiration (physiological acclimation) or reduced construction cost of stem tissue (structural acclimation). Maintenance respiration per unit sapwood volume at 15°C did not differ between populations (desert: 6.39 ± 1.14 SE μmol m⁻³ s⁻¹, montane: 6.54 ± 1.13 SE μmol m⁻³ s⁻¹, P = 0.71) and declined with increasing stem diameter (P = 0.001). The temperature coefficient of respiration (Q ₁₀) varied seasonally within both environments (P = 0.05). Construction cost of stem sapwood was the same in both environments (desert: 1.46 ± 0.009 SE g glucose g⁻¹ sapwood, montane: 1.48 ± 0.009 SE glucose g⁻¹ sapwood, P = 0.14). Annual construction respiration calculated from construction cost, percent carbon and relative growth rate was greater in montane populations due to higher growth rates. These data provide no evidence of respiratory acclimation by desert trees. Estimated yearly stem maintenance respiration was greater in large desert trees than in large montane trees because of higher temperatures in the desert and because of increased allocation of biomass to sapwood. By analogy, these data suggest that under predicted increases in temperature and aridity, potential increases in aboveground carbon gain due to enhanced photosynthetic rates may be partially offset by increases in maintenance respiration in large trees growing in CO₂-enriched atmospheres. Received: 4 November 1996 / Accepted: 23 January 1997     

Ferric reduction by iron-limited Chlamydomonas cells interacts with both photosynthesis and respiration

Iron limitation led to a large increase in extracellular ferricyanide (Fe[III]) reductase activity in cells of the green alga Chlamydomonas reinhardtii Dangeard. Mass-spectrometric measurement of gas exchange indicated that ferricyanide reduction in the dark resulted in a stimulation of respiratory CO₂ production without affecting the rate of respiratory O₂ consumption, consistent with the previously postulated activation of the oxidative pentose phosphate pathway in support of Fe(III) reduction by iron-limited Chlamydomonas cells (X. Xue et al., 1998, J. Phycol. 34: 939–944). At saturating irradiance, the rate of ferricyanide reduction was stimulated almost 3-fold, and this stimulation was inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. Ferricyanide reduction during photosynthesis resulted in approximately a 50% inhibition of photosynthetic CO₂ fixation at saturating irradiance, and almost 100% inhibition of CO₂ fixation at sub-saturating irradiance. Photosynthesis by iron-sufficient cells was not affected by ferricyanide addition. Addition of 250 μM ferricyanide to iron-limited cells in which photosynthesis was inhibited (either by the presence of glycolaldehyde, or by maintaining the cells at the CO₂ compensation point) resulted in a stimulation in the rate of gross photosynthetic O₂ evolution. Chlorophyll a fluorescence measurements indicated a large increase in non-photochemical quenching during ferricyanide reduction in the light; the increase in nonphotochemical quenching was abolished by the addition of nigericin. These results suggest that reduction of extracellular ferricyanide (mediated at the plasma membrane) interacts with both photosynthesis and respiration, and that both of these processes contribute NADPH in the light. Received: 15 September 1999 / Accepted: 14 October 1999     

Influence of stand structure on carbon-13 of vegetation, soils, and canopy air within deciduous and evergreen forests in Utah, United States

Carbon isotope ratios (δ¹³C) were studied in evergreen and deciduous forest ecosystems in semi-arid Utah (Pinus contorta, Populus tremuloides, Acer negundo and Acer grandidentatum). Measurements were taken in four to five stands of each forest ecosystem differing in overstory leaf area index (LAI) during two consecutive growing seasons. The δ¹³C_leaf (and carbon isotope discrimination) of understory vegetation in the evergreen stands (LAI 1.5–2.2) did not differ among canopies with increasing LAI, whereas understory in the deciduous stands (LAI 1.5–4.5) exhibited strongly decreasing δ¹³C_leaf values (increasing carbon isotope discrimination) with increasing LAI. The δ¹³C values of needles and leaves at the top of the canopy were relatively constant over the entire LAI range, indicating no change in intrinsic water-use efficiency with overstory LAI. In all canopies, δ¹³C_leaf decreased with decreasing height above the forest floor, primarily due to physiological changes affecting c _i/c _a (> 60%) and to a minor extent due to δ¹³C of canopy air (< 40%). This intra-canopy depletion of δ¹³C_leaf was lowest in the open stand (1‰) and greatest in the denser stands (4.5‰). Although overstory δ¹³C_leaf did not change with canopy LAI, δ¹³C of soil organic carbon increased with increasing LAI in Pinus contorta and Populus tremuloides ecosystems. In addition, δ¹³C of decomposing organic carbon became increasingly enriched over time (by 1.7–2.9‰) for all deciduous and evergreen dry temperate forests. The δ¹³C_canopy of CO₂ in canopy air varied temporally and spatially in all forest stands. Vertical canopy gradients of δ¹³C_canopy, and [CO₂]_canopy were larger in the deciduous Populus tremuloides than in the evergreen Pinu contorta stands of similar LAI. In a very wet and cool year, ecosystem discrimination (Δ_e) was similar for both deciduous Populus tremulodies (18.0 ± 0.7‰) and evergreen Pinus contorta (18.3 ± 0.9‰) stands. Gradients of δ¹³C_canopy and [CO₂]_canopy were larger in denser Acer spp. stands than those in the open stand. However, ¹³C enrichment above and photosynthetic draw-down of [CO₂]_canopy below tropospheric baseline values were larger in the open than in the dense stands, due to the presence of a vigorous understory vegetation. Seasonal patterns of the relationship δ¹³C_canopy versus 1/[CO₂]_canopy were strongly influenced by precipitation and air temperature during the growing season. Estimates of Δ_e for Acer spp. did not show a significant effect of stand structure, and averaged 16.8 ± 0.5‰ in 1933 and 17.4 ± 0.7‰ in 1994. However, Δ_e varied seasonally with small fluctuations for the open stand (2‰), but more pronounced changes for the dense stand (5‰). Received: 15 April 1996 / Accepted: 19 October 1996     

Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures

Maize (Zea mays L.) cell cultures incorporated radioactivity from [¹⁴C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the CoA pool had lost its ¹⁴C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1–3 d) cultures, polysaccharide-bound [¹⁴C]feruloyl- and [¹⁴C]diferuloyl residues were both detectable within 1 min of [¹⁴C]cinnamate feeding. Thus, feruloyl residues were dimerised <1 min after their attachment to polysaccharides. For at least the first 2.3 h after [¹⁴C]cinnamate feeding, polysaccharide-bound [¹⁴C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides <1 min after the biosynthesis of the latter, and >10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H₂O₂ (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [¹⁴C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H₂O₂ induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H₂O₂-limited. In both 2- and 4-d-old cultures, polysaccharide-bound ¹⁴C-trimers and larger coupling products exceeded [¹⁴C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed. Received: 19 January 2000 / Accepted: 13 April 2000     

Light-dependent bicarbonate uptake and CO2 efflux in the marine microalga Nannochloropsis gaditana

 Inorganic carbon (Ci) uptake and efflux has been investigated in the marine microalga Nannochloropsis gaditana Lubian by monitoring CO₂ fluxes in cell suspensions using mass spectrometry. Addition of H¹³CO₃ ⁻ to cell suspensions in the dark caused a transient increase in the CO₂ concentration in the medium far in excess of the equilibrium CO₂ concentration. The magnitude of this release was dependent on the length of time the cells had been kept in the dark. Once equilibrium between the Ci species had been achieved, a CO₂ efflux was observed after saturating light intensity was applied to the cells. External carbonic anhydrase (CA) was not detected nor does this species demonstrate a capacity to take up CO₂ by active transport. Photosynthetic O₂ evolution and the release CO₂ in the dark depend on HCO₃ ⁻ uptake since both were inhibited by the anion exchange inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). The bicarbonate uptake mechanism requires light but can also continue for short periods in the dark. Ethoxyzolamide, a CA inhibitor, markedly inhibited CO₂ efflux in the dark, indicating that CO₂ efflux was dependent upon the intracellular dehydration of HCO₃ ⁻. These results indicate that Nannochloropsis possesses a bicarbonate uptake system which causes the accumulation of high intracellular Ci levels and an internal CA which maintains the equilibrium between CO₂ and HCO₃ ⁻ and thus causes a subsequent release of CO₂ to the external medium. Received: 20 September 1999 / Accepted: 25 October 1999     

Affinity for inorganic carbon of Gracilaria tenuistipitata cultured at low and high irradiance

Regulation by irradiance level of the mechanism for dissolved inorganic carbon (DIC) acquisition was examined in the red macroalga Gracilaria tenuistipitata Zhang et Xia. For this purpose, affinity for external DIC, carbonic anhydrase (CA; EC 4.2.1.1) activity and content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) were determined in thalli grown at 45 and 500 μmol photons m⁻² s⁻¹. Oxygen evolution rates declined by 50% when the medium pH was changed from 8.1 to 8.7, and the pH compensation point attained was ca. 9.2. These characteristics were unaffected by the light treatments. In contrast, photosynthetic conductance for DIC at pH 8.7 was doubled in thalli grown at high irradiance compared with those grown at low irradiance (to 0.74 × 10⁻⁶ from 0.33 × 10⁻⁶ m s⁻¹). Photosynthetic rates at saturating DIC concentration were also higher by 60% in thalli grown at high irradiance. These differences could not be attributed to changes in the use of external DIC, since external CA activity did not vary. Although the irradiance level did not modify the pool size of Rubisco, Rubisco content expressed on a chlorophyll a basis was almost doubled at high irradiance. These results likely indicate that the internal transport of DIC towards the active-site of Rubisco, rather than the external use of DIC, is enhanced in the thalli grown at high irradiance. Received: 7 June 1999 / Accepted: 16 October 1999     

Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm

The dye FM1-43 was used alone or in combination with measurements of the membrane capacitance (C_m) to monitor membrane changes in protoplasts from Viciafaba L. guard cells. Confocal images of protoplasts incubated with FM1-43 (10 μM) at constant ambient osmotic pressure (π_o) revealed in confocal images a slow internalisation of FM1-43-labelled membrane into the cytoplasm. As a result of this process the relative fluorescence intensity of the cell interior (f_FM,i) increased with reference to the total fluorescence (f_FM,t) by 7.4 × 10⁻⁴ min⁻¹. This steady internalisation of dye suggests the occurrence of constitutive endocytosis under constant osmotic pressure. Steady internalisation of FM1-43 labelled membrane caused a prominent staining of a ring-like structure located beneath the plasma membrane. Abrupt elevation of π_o by 200 mosmol kg⁻¹ caused, over the first minutes of incubation, a rapid internalisation of FM1-43 fluorescence into the cytoplasm concomitant with a decrease in cell perimeter. Within the first 5 min the cell perimeter decreased by 7.9%. Over the same time f_FM,i/f_FM,t increased by 0.13, reflecting internalisation of fluorescent label into the cytoplasm. Combined measurements of C_m and total fluorescence of a protoplast (f_FM,p) showed that an increase in π_o evoked a decrease in C_m but no change in f_FM,p. This means that surface contraction of the protoplast is due to retrieval of excess membrane from the plasma membrane and internalisation into the cytoplasm. Further inspection of confocal images revealed that protoplast shrinking was only occasionally associated with internalisation of giant vesicles (median diameter 2.7 μm) with FM1-43-labelled membrane. But, in all cases, osmotic contraction was correlated with a diffuse distribution of FM1-43 label throughout the cytoplasm. From this, we conclude that endocytosis of small vesicles into the cytoplasm is the obligatory process by which cells accommodate an osmotically driven decrease in membrane surface area. Received: 4 May 1999 / Accepted: 19 August 1999     

Stability of metal ion complexes formed with methyl phosphate and hydrogen phosphate

 The acidity constants of methyl phosphoric acid, CH₃OPO(OH)₂, and orthophosphoric acid, HOPO(OH)₂, and the stability constants of the 1 : 1 complexes formed between Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, or Cd²⁺ and methyl phosphate, CH₃OPO₃ ^2–, or hydrogen phosphate, HOPO₃ ^2–, were determined by potentiometric pH titration in aqueous solution (25  °C;I = 0.1 M, NaNO₃). On the basis of previously established log K versus pK _a straight-line plots for the complexes of simple phosphate monoesters and phosphonate derivatives, R-PO₃ ^2–, where R is a noncoordinating residue, it is shown that the stability of the M(CH₃OPO₃) complexes is solely determined (as one might expect) by the basicity of the –PO₃ ^2– residue. It is emphasized that the mentioned reference lines may also be used to reveal increased complex stabilities, for example, for certain complexes formed with 8-quinolyl phosphate the occurrence of 7-membered chelates can be proven in this way; the same procedure is also applicable to complexes of nucleotides, etc. The M(HOPO₃) complexes are slightly more stable (on average by 0.08 log unit) than it is expected from the basicity of HPO₄ ^2–; this observation is attributed to a more effective solvation, including hydrogen bonding, than is possible with CH₃OPO₃ ^2– species. Received: 9 November 1995 / Accepted: 5 February 1996