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     

The role of external carbonic anhydrase in the photosynthetic use of inorganic carbon in the deep-water alga Phyllariopsis purpurascens (Laminariales, Phaeophyta)

Mechanisms of inorganic carbon assimilation were investigated in the deep-water alga Phyllariopsis purpurascens (C. Agardh) Henry et South (Laminariales, Phaeophyta). The gross photosynthetic rate as a function of external pH, at a constant concentration of 2 mM dissolved inorganic carbon (DIC), decreased sharply from pH 7.0 to 9.0, and was not substantially different from 0 above pH 9.0. These data indicate that P. purpurascens is inefficient in the use of external HCO₃ ⁻ as a carbon source in photosynthesis. Moreover, the photosynthetic rate as a function of external DIC and the highest pH (9.01 ± 0.07) that this species can achieve in a closed system were consistent with a low capacity to use HCO₃ ⁻, in comparison to many other species of seaweeds. The role of external carbonic anhydrase (CA; EC 4.2.1.1) on carbon uptake was investigated by measuring both the HCO₃ ⁻-dependent O₂ evolution and the CO₂ uptake, at pH 5.5 and 8.0, and the rate of pH change in the external medium, in the presence of selected inhibitors of extra- and intracellular CA. Photosynthetic DIC-dependent O₂ evolution was higher at pH 5.5 (where CO₂ is the predominant form of DIC) than at pH 8.0 (where the predominant chemical species is HCO₃ ⁻). Both intra- and extracellular CA activity was detected. Dextran-bound sulfonamide (DBS; a specific inhibitor of extracellular CA) reduced the photosynthetic O₂ evolution and CO₂ uptake at pH 8.0, but there was no effect at pH 5.5. The pH-change rate of the medium, under saturating irradiance, was reduced by DBS. Phyllariopsis purpurascens has a low efficiency in the use of HCO₃ ⁻ as carbon source in photosynthesis; nevertheless, the ion can be used after dehydration, in the external medium, catalyzed by extracellular CA. This mechanism could explain why the photosynthetic rate in situ was higher than that supported solely by the diffusion of CO₂ from seawater. Received: 6 March 1998 / Accepted: 22 June 1998     

Artificially increased ascorbate content affects zeaxanthin formation but not thermal energy dissipation or degradation of antioxidants during cold-induced photooxidative stress in maize leaves

Infiltrating detached maize (Zeamays L.) leaves with L-galactono-1,4-lactone (L-GAL) resulted in a 4-fold increase in the content of leaf ascorbate. Upon exposure to high irradiance (1000 μmol photons m⁻² s⁻¹) at 5 °C, L-GAL leaves de-epoxidized the xanthophyll-cycle pigments faster than the control leaves; the maximal ratio of de-epoxidized xanthophyll-cycle pigments to the whole xanthophyll-cycle pool was the same in both leaf types. The elevated ascorbate content, together with the faster violaxanthin de-epoxidation, did not affect the degree of photoinhibition and the kinetics of the recovery from photoinhibition, assayed by monitoring the maximum quantum efficiency of photosystem II primary photochemistry (F_v/F_m). Under the experimental conditions, the thermal energy dissipation seems to be zeaxanthin-independent since, in contrast to the de-epoxidation, the decrease in the efficiency of excitation-energy capture by open photosystem II reaction centers (F_v′/F_m′) during the high-irradiance treatment at low temperature showed the same kinetic in both leaf types. This was also observed for the recovery of the maximal fluorescence after stress. Furthermore, the elevated ascorbate content did not diminish the degradation of pigments or α-tocopherol when leaves were exposed for up to 24 h to high irradiance at low temperature. Moreover, a higher content of ascorbate appeared to increase the requirement for reduced glutathione. Received: 20 May 1999 / Accepted: 29 October 1999     

Mechanisms of inorganic carbon acquisition in Gracilaria gaditana nom. prov. (Rhodophyta)

The mechanisms for acquisition of dissolved inorganic carbon (DIC) in the red macroalga Gracilaria gaditana nom. prov. have been investigated. The capacity for HCO₃ ⁻ use by an extracellular carbonic anhydrase (CA; EC 4.2.1.1), and by an anion exchanger with similar properties to that of red blood cells (AE1), has been quantified. It was illustrated by comparing O₂ evolution rates with those theoretically supported by CO₂, as well as by photosynthesis-pH curves. Both external and internal CA, and a direct uptake were involved in HCO₃ ⁻ use, since photosynthesis and pH evolution were affected by acetazolamide, 6-ethoxyzolamide (inhibitors of external and total CA, respectively) and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate, (DIDS; an inhibitor of HCO₃ ⁻ exchanger protein). The activity of the external CA was detected by a potentiometric method and by an alternative method based on the study of O₂ evolution after addition of CO₂ and acetazolamide. The latter method showed a residual photosynthetic rate due to direct HCO₃ ⁻ use. Inhibitors caused a reduction in the pH compensation points in pH-drift experiments. The CO₂ compensation points for photosynthesis increased when the inhibitors were applied, indicating a suppresion of the pathways involved in the carbon-concentrating mechanism. The net photosynthesis rates as a function of DIC concentration displayed a biphasic pattern that could be supported by the occurrence of the two mechanisms of HCO₃ ⁻ use. The potential contribution to HCO₃ ⁻ acquisition by the DIDS-sensitive mechanism was higher after culturing at a high pH. Our results suggest that the HCO₃ ⁻ use by Gracilaria gaditana is carried out by the two DIC uptake mechanisms. These operate simultaneously with different affinities for DIC, the indirect HCO₃ ⁻ use by an external CA activity being the main pathway. The presence of a carbon-concentrating mechanism confers eco-physiological advantages in a fluctuating ecosystem subjected daily to high pHs and low DIC concentrations. Received: 3 July 1998 / Accepted: 30 November 1998     

Leaf photosynthesis, plant growth and nitrogen allocation in rice under different irradiances

The photosynthetic rates and various components of photosynthesis including ribulose-1,5-bisphosphate carboxylase (Rubisco; EC 4.1.1.39), chlorophyll (Chl), cytochrome (Cyt) f, and coupling factor 1 (CF₁) contents, and sucrose-phosphate synthase (SPS; EC 2.4.1.14) activity were examined in young, fully expanded leaves of rice (Oryza sativa L.) grown hydroponically under two irradiances, namely, 1000 and 350 μmol quanta · m⁻² · s⁻¹, at three N concentrations. The light-saturated rate of photosynthesis measured at 1800 μmol · m⁻² · s⁻¹ was almost the same for a given leaf N content irrespective of growth irradiance. Similarly, Rubisco content and SPS activity were not different for the same leaf N content between irradiance treatments. In contrast, Chl content was significantly greater in the plants grown at 350 μmol · m⁻² · s⁻¹, whereas Cyt f and CF₁ contents tended to be slightly smaller. However, these changes were not substantial, as shown by the fact that the light-limited rate of photosynthesis measured at 350 μmol · m⁻² · s⁻¹ was the same or only a little higher in the plants grown at 350 μmol · m⁻² · s⁻¹ and that CO₂-saturated photosynthesis did not differ between irradiance treatments. These results indicate that growth-irradiance-dependent changes in N partitioning in a leaf were far from optimal with respect to N-use efficiency of photosynthesis. In spite of the difference in growth irradiance, the relative growth rate of the whole plant did not differ between the treatments because there was an increase in the leaf area ratio in the low-irradiance-grown plants. This increase was associated with the preferential N-investment in leaf blades and the extremely low accumulation of starch and sucrose in leaf blades and sheaths, allowing a more efficient use of the fixed carbon. Thus, morphogenic responses at the whole-plant level may be more important for plants as an adaptation strategy to light environments than a response of N partitioning at the level of a single leaf. Received: 23 February 1997 / Accepted: 8 May 1997     

Acclimation of the summer annual species, Lolium temulentum, to CO2 enrichment

Lolium temulentum L. Ba 3081 was grown hydroponically in air (350 μmol mol⁻¹ CO₂) and elevated CO₂ (700 μmol mol⁻¹ CO₂) at two irradiances (150 and 500 μmol m⁻² s⁻¹) for 35 days at which point the plants were harvested. Elevated CO₂ did not modify relative growth rate or biomass at either irradiance. Foliar carbon-to-nitrogen ratios were decreased at elevated CO₂ and plants had a greater number of shorter tillers, particularly at the lower growth irradiance. Both light-limited and light-saturated rates of photosynthesis were stimulated. The amount of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) protein was increased at elevated CO₂, but maximum extractable Rubisco activities were not significantly increased. A pronounced decrease in the Rubisco activation state was found with CO₂ enrichment, particularly at the higher growth irradiance. Elevated-CO₂-induced changes in leaf carbohydrate composition were small in comparison to those caused by changes in irradiance. No CO₂-dependent effects on fructan biosynthesis were observed. Leaf respiration rates were increased by 68% in plants grown with CO₂ enrichment and low light. We conclude that high CO₂ will only result in increased biomass if total light input favourably increases the photosynthesis-to-respiration ratio. At low irradiances, biomass is more limited by increased rates of respiration than by CO₂-induced enhancement of photosynthesis. Received: 23 February 1999 / Accepted: 15 June 1999     

Assessment of optimal depth and photon irradiance for cultivation of the brown alga, <Emphasis Type="Italic">Sargassum fulvellum</Emphasis> (Turner) C. Agardh

This study was aimed at determining the optimal depth and photon irradiance for growth of Sargassum fulvellum. Sampling and measurement of underwater irradiance were carried out at farms cultivating S. fulvellum at Wando, southwestern coast of Korea, from May 2004 to April 2005. Growth of thalli, underwater irradiance and photosynthetic quantum yield were measured over a range of depths for three culture stages. During their nursery cultivation stage (Stage I), length increase was greatest at 1.5 m depth (2.5 ± 0.2 cm), where the average midday irradiance over 28 days was 488 ± 58 μmol photons m⁻² s⁻¹. During the pre-main cultivation stage (Stage II), the greatest length increase occurred at 1 m depth (10.9 ± 0.1 cm) with an average irradiance of 845 ± 169 μmol photons m⁻² s⁻¹. For the main cultivation stage (Stage III) of the alga, thalli showed maximal length growth in March and early April at depths of 1–2 m and 3 m. These results suggest that growth at each cultivation stage of S. fulvellum could be controlled by depth of cultivation rope. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Regulation of the mechanism for HCO 3 − use by the inorganic carbon level in Porphyra leucosticta Thur. in Le Jolis (Rhodophyta)

The capacity for HCO₃ ⁻ use by Porphyra leucosticta Thur. in Le Jolis grown at different concentrations of inorganic carbon (C_i) was investigated. The use of HCO₃ ⁻ at alkaline pH by P. leucosticta was␣demonstrated by comparing the O₂ evolution rates measured with the O₂ evolution rates theoretically supported by the CO₂ spontaneously formed from HCO₃ ⁻ . Both external and internal carbonic anhydrase (CA; EC 4.2.1.1) were implied in HCO₃ ⁻ use during photosynthesis because O₂ evolution rates and the increasing pH during photosynthesis were inhibited in the presence of azetazolamide and ethoxyzolamide (inhibitors for external and total CA respectively). Both external and internal CA were regulated by the C_i level at which the algae were grown. A high C_i level produced a reduction in total CA activity and a low C_i level produced an increase in total CA activity. In contrast, external CA was increased at low C_i although it was not affected at high C_i . Parallel to the reduction in total CA activity at high C_i is a reduction in the affinity for C_i, as estimated from photosynthesis versus C_i curves, was found. However, there was no evident relationship between external CA activity and the capacity for HCO₃ ⁻ use because the presence of external CA became redundant when P. leucosticta was cultivated at high C_i. Our results suggest that the system for HCO₃ ⁻ use in P. leucosticta is composed of different elements that can be activated or inactivated separately. Two complementary hypotheses are postulated: (i) internal CA is an absolute requirement for a functioning C_i-accumulation mechanism; (ii) there is a CO₂ transporter that works in association with external CA. Received: 20 April 1996 / Accepted: 5 August 1996     

Cultivation of the green alga, Codium fragile (Suringar) Hariot, by artificial seed production in Korea

Codium fragile (Suringar) Hariot is an edible green alga farmed in Korea using seed stock produced from regeneration of isolated utricles and medullary filaments. Experiments were conducted to reveal the optimal conditions for nursery culture and out-growing of C. fragile. Sampling and measurement of underwater irradiance were carried out at farms cultivating C. fragile at Wando, on the southwestern coast of Korea, from October 2004 to August 2005. Growth of erect thalli and underwater irradiance were measured over a range of depths for three culture stages. During the nursery cultivation stage (Stage I), growth rate was greatest at 0.5 m depth (0.055 ± 0.032 mm day⁻¹), where the average midday irradiance over 60 days was 924 ± 32 μmol photons m⁻² s⁻¹. During the pre-main cultivation stage (Stage II), the greatest growth rate occurred at a depth of 2 m (0.113 ± 0.003 mm day⁻¹) with an average irradiance of 248 ± 116 μmol photons m⁻² s⁻¹. For the main cultivation stage (Stage III) of the alga, thalli achieved the greatest increase in biomass at 1 m depth (7.2 ± 1.0 kg fresh wt m⁻¹). These results suggest that optimal growth at each cultivation stages of C. fragile could be controlled by depth of cultivation rope.     

Inoculation and nitrate alter phytohormone levels in soybean roots: differences between a supernodulating mutant and the wild type

 The levels of different cytokinins, indole-3-acetic acid (IAA) and abscisic acid (ABA) in roots of Glycine max [L.] Merr. cv. Bragg and its supernodulating mutant nts382 were compared for the first time. Forty-eight hours after inoculation with Bradyrhizobium, quantitative and qualitative differences were found in the root's endogenous hormone status between cultivar Bragg and the mutant nts382. The six quantified cytokinins, ranking similarly in each genotype, were present at higher concentrations (30–196% on average for isopentenyl adenosine and dihydrozeatin riboside, respectively) in mutant roots. By contrast, the ABA content was 2-fold higher in Bragg, while the basal levels of IAA [0.53 μmol (g DW)⁻¹, on average] were similar in both genotypes. In 1 mM NO₃ ⁻-fed Bragg roots 48 h post-inoculation, IAA, ABA and the cytokinins isopentenyl adenine, and isopentenyl adenosine quantitatively increased with respect to uninoculated controls. However, only the two cytokinins increased in the mutant. High NO₃ ⁻ (8 mM) markedly reduced root auxin concentration, and neither genotypic differences nor the inoculation-induced increase in auxin concentration in Bragg was observed under these conditions. Cytokinins and ABA, on the other hand, were little affected by 8 mM NO₃ ⁻. Root IAA/cytokinin and ABA/cytokinin ratios were always higher in Bragg relative to the mutant, and responded to inoculation (mainly in Bragg) and nitrate (both genotypes). The overall results are consistent with the auxin-burst-control hypothesis for the explanation of autoregulation and supernodulation in soybean. However, they are still inconclusive with respect to the inhibitory effect of NO₃ ⁻. Received: 16 April 1999 / Accepted: 13 December 1999     

Intracellular chloroplast photorelocation in the moss Physcomitrella patens is mediated by phytochrome as well as by a blue-light receptor

 The light-induced intracellular relocation of chloroplasts was examined in red-light-grown protonemal cells of the moss Physcomitrella patens. When irradiated with polarized red or blue light, chloroplast distribution in the cell depended upon the direction of the electrical vector (E-vector) in both light qualities. When the E-vector was parallel to the cross-wall (i.e. perpendicular to the protonemal axis), chloroplasts accumulated along the cross-wall; however, no accumulation along the cross-wall was observed when the E-vector was perpendicular to it (i.e. parallel to the protonemal axis). When a part of the cell was irradiated with a microbeam of red or blue light, chloroplasts accumulated at or avoided the illumination point depending on the fluence rate used. Red light of 0.1–18 W m⁻² and blue light of 0.01–85.5 W m⁻² induced an accumulation response (low-fluence-rate response; LFR), while an avoidance response (high-fluence-rate response; HFR) was induced by red light of 60 W m⁻² or higher and by blue light of 285 W m⁻². The red-light-induced LFR and HFR were nullified by a simultaneous background irradiation of far-red light, whereas the blue-light-induced LFR and HFR were not affected at all by this treatment. These results show, for the first time, that dichroic phytochrome, as well as the dichroic blue-light receptor, is involved in the chloroplast relocation movement in these bryophyte cells. Further, the phytochrome-mediated responses but not the blue-light responses were revealed to be lost when red-light-grown cells were cultured under white light for 2 d. Received: 7 September 1999 / Accepted: 15 October 1999     

The application of atomic force microscopy to topographical studies and force measurements on the secreted adhesive of the green alga Enteromorpha

Atomic force microscopy (AFM) enables the topographical structure of cells and biological materials to be resolved under natural (physiological) conditions, without fixation and dehydration artefacts associated with imaging methods in vacuo. It also provides a means of measuring interaction forces and the mechanical properties of biomaterials. In the present study, AFM has been applied for the first time to the study of the mechanical properties of a natural adhesive produced by a green plant cell. Swimming spores of the green alga Enteromorpha linza (L.) J. Ag. (7–10 μm) secrete an adhesive glycoprotein which provides firm anchorage to the substratum. Imaging of the adhesive in its hydrated state revealed a swollen gel-like pad, approximately 1 μm thick, surrounding the spore body. Force measurements revealed that freshly released adhesive has an adhesion strength of 173 ± 1.7 mN m⁻¹ (mean ± SE; n=90) with a maximum value for a single adhesion force curve of 458 mN m⁻¹. The adhesive had a compressibility (equivalent to Young's modulus) of 0.54 × 10⁶ ± 0.05 × 10⁶ N m⁻² (mean ± SE; n=30). Within minutes of release the adhesive underwent a progressive `curing' process with a 65% reduction in mean adhesive strength within an hour of settlement, which was also reflected in a reduction in the average length of the adhesive polymer strands (polymer extension) and a 10-fold increase in Young's modulus. Measurements on the spore surface itself revealed considerably lower adhesion-strength values but higher polymer-extension values than the adhesive pad, which may reflect the deposition of different polymers on this surface as a new cell wall is formed. The study demonstrates the value of AFM to the imaging of plant cells in the absence of fixation and dehydration artefacts and to the characterisation of the mechanical properties of plant glycoproteins that have potential utility as adhesives. Received: 22 February 2000 / Accepted: 20 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     

Body fluid homeostasis and cardiovascular adjustments during submaximal exercise: influence of chewing coca leaves

 The present study was undertaken to determine the haematological and cardiovascular status, at rest and during prolonged (1 h) submaximal exercise (approximately 70% of peak oxygen uptake) in a group (n = 12) of chronic coca users after chewing approximately 50 g of coca leaves. The results were compared to those obtained in a group (n = 12) of nonchewers. At rest, coca chewing was accompanied by a significant increase in heart rate [from 60 (SEM 4) TO 76 (SEM 3) beats · min⁻¹], in haematocrit [from 53.2 (SEM 1.2) to 55.6 (SEM 1.1)%] in haemoglobin concentration, and plasma noradrenaline concentration [from 2.8 (SEM 0.4) to 5.0 (SEM 0.5) μmol · l⁻¹]. It was calculated that coca chewing for 1 h resulted in a significant decrease in blood [−4.3 (SEM 2.2)%] and plasma [−8.7 (SEM 1.2)%] volume. During submaximal exercise, coca chewers displayed a significantly higher heart rate and mean arterial blood pressure. The exercise-induced haemoconcentration was blunted in coca chewers compared to nonchewers. It was concluded that the coca-induced fluid shift observed at rest in these coca chewers was not cumulative with that of exercise, and that the hypovolaemia induced by coca chewing at rest compromised circulatory adjustments during exercise. Accepted: 29 October 1996     

Irradiance prior to and during desiccation improves the tolerance to excess irradiance in the desiccated state of the old forest lichen Lobaria pulmonaria

Hydrated thalli of the lichen Lobaria pulmonaria were either preconditioned to dim irradiance (DI, 5 μmol m⁻² s⁻¹) or medium irradiance (MI, 200 μmol m⁻² s⁻¹) for 6 h. After this 6 h period, the thalli were allowed to desiccate under the two respective irradiances. Thereafter, these dry lichens were exposed to high irradiance (HI, 1 000 μmol m⁻² s⁻¹) for 60 h. After this HI treatment, the maximal photochemical quantum yield (F_V/F_M) and the de-epoxidation state of xanthophyll cycle pigments (DEPS) were highest in thalli preconditioned to MI. Hence irradiance in the last hydrated period before sampling is significant for the physiological state of lichens. A standardized irradiance pre-treatment before start of experiments is recommended.     

Variations in leaf β13C along a vertical profile of irradiance in a temperate Japanese forest

The vertical profile of stable carbon isotope ratios (δ¹³C) of leaves was analyzed for 13 tree species in a cool-temperate deciduous forest in Japan. The vertical distribution of long-term averaged δ¹³C in atmospheric CO₂ (δ_a) was estimated from δ¹³C of dry matter from NADP-malic enzyme type C₄ plant (Zea mays L. var. saccharata Sturt.) grown at a tower in the forest for 32␣days, assuming constant Δ value (3.3‰) in Z. mays against height. The δ_a value obtained from δ¹³C in Z.␣mays was lowest at the forest floor (−9.30 ± 0.03‰), increased with height, and was almost constant above 10␣m (−7.14 ± 0.14‰). Then leaf Δ values for the tree species were calculated from tree leaf δ¹³ C andδ_a. Mean leaf Δ values for the three tall deciduous species (Fraxinus mandshurica, Ulmus davidiana, and Alnus hirsuta) were significantly different among three height levels in the forest: 23.1 ± 0.7‰ at the forest floor (understory), 21.4 ± 0.5‰ in lower canopy, and 20.5 ± 0.3‰ in upper canopy. The true difference in tree leaf Δ among the forest height levels might be even greater, because Δ in Z. mays probably increased with shading by up to ∼‰. The difference in tree leaf Δ among the forest height levels would be mainly due to decreasing intercellular CO₂ (C _i) with the increase in irradiance. Potential assimilation rate for the three tree species probably increased with height, since leaf nitrogen content on an area basis for these species also increased with height. However, the increase in stomatal conductance for these tree species would fail to meet the increase in potential assimilation rate, which might lead to increasing the degree of stomatal limitation in photosynthesis with height. Received: 30 September 1995 / Accepted: 25 October 1996     

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     

A CO2-Flux Mechanism Operating via pH-Polarity in Hydrilla Verticillata Leaves With C3 and C4 Photosynthesis

The aquatic angiosperm Hydrilla verticillata lacks Kranz anatomy, but has an inducible, C₄-based, CO₂ concentrating mechanism (CCM) that concentrates CO₂ in the chloroplasts. Both C₃ and C₄ Hydrilla leaves showed light-dependent pH polarity that was suppressed by high dissolved inorganic carbon (DIC). At low DIC (0.25 mol m⁻³), pH values in the unstirred water layer on the abaxial and adaxial sides of the leaf were 4.2 and10.3, respectively. Abaxial apoplastic acidification served as a CO₂ flux mechanism (CFM), making HCO₃ ⁻ available for photosynthesis by conversion to CO₂. DIC at 10 mol m⁻³ completely suppressed acidification and alkalization. The data, along with previous results, indicated that inhibition was specific to DIC, and not a buffer effect. Acidification and alkalization did not necessarily show 1:1 stoichiometry; their kinetics for the apolar induction phase differed, and alkalization was less inhibited by 2.5 mol m⁻³ DIC. At low irradiance (50 μmol photons m⁻² s⁻¹), where CCM activity in C₄ leaves is minimized, both leaf types had similar DIC inhibition of pH polarity. However, as irradiance increased, DIC inhibition of C₃ leaves decreased. In C₄ leaves the CFM and CCM seemed to compete for photosynthetic ATP and/or reducing power. The CFM may require less, as at low irradiance it still operated maximally, if [DIC] was low. Iodoacetamide (IA), which inhibits CO₂ fixation in Hydrilla, also suppressed acidification and alkalization, especially in C₄ leaves. IA does not inhibit the C₄ CCM, which suggests that the CFM and CCM can operate independently. It has been hypothesized that irradiance and DIC regulate pH polarity by altering the chloroplastic [DIC], which effects the chloroplast redox state and subsequently redox regulation of a plasma-membrane H⁺-ATPase. The results lend partial support to a down-regulatory role for high chloroplastic [DIC], but do not exclude other sites of DIC action. IA inhibition of pH polarity seems inconsistent with the chloroplast NADPH/NADP⁺ ratio being the redox transducer. The possibility that malate and oxaloacetate shuttling plays a role in CFM regulation requires further investigation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inactivation of DNA replication origins by the cell cycle regulator, trigonelline, in root meristems of Lactuca sativa

 The effects of trigonelline (TRG) on the cell cycle in root meristems of Lactuca sativa L. were examined in the knowledge that TRG is a cell cycle regulator that causes cell arrest in G2, and prevents ligation of replicons in S-phase. The hypothesis was tested that continuous exposure to TRG would perturb DNA replication which, in turn, would lengthen the cell cycle and impair root elongation. Using DNA fibre autoradiography, mean replicon size was 31 and 13 μm in the TRG (3 mM) and control treatments, respectively. Trigonelline also resulted in a lengthening of both S-phase and the cell cycle and a decrease in primary root elongation. Hence, replicon inactivation was responsible for the protracted S-phase. Trigonelline treatment also resulted in a 1.6-fold increase in fork rate (13.8 μm h⁻¹) compared with the control (8.4 m h⁻¹). The faster fork rate in the larger replicons is in accord with the highly significant positive relationship already established between fork rate and replicon size for various unrelated higher plants. Received: 11 October 1999 / Accepted: 23 December 1999     

Short latency vestibular evoked potentials in the Japanese quail (Coturnix coturnix japonica)

Short-latency vestibular-evoked potentials to pulsed linear acceleration were characterized in the quail. Responses occurred within 8 ms following the onset of stimuli and were composed of a series of positive and negative peaks. The latencies and amplitudes of the first four peaks were quantitatively characterized. Mean latencies at 1.0 g ms⁻¹ ranged from 1265 ± 208 μs (P1, N = 18) to 4802 ± 441 μs (N4, N = 13). Amplitudes ranged from 3.72 ± 1.51 μV (P1/N1, N = 18) to 1.49 ± 0.77 μV (P3/N3, N = 16). Latency-intensity (LI) slopes ranged from −38.7 ± 7.3 μs dB⁻¹ (P1, N = 18) to −71.6 ± 21.9 μs dB⁻¹ (N3, N = 15) and amplitude-intensity (AI) slopes ranged from 0.20 ± 0.08 μV dB⁻¹ (P1/N1, N = 18) to 0.07 ± 0.04 μV dB⁻¹ (P3/N3, N = 11). The mean response threshold across all animals was −21.83 ± 3.34 dB re: 1.0 g ms⁻¹ (N = 18). Responses remained after cochlear extirpation showing that they could not depend critically on cochlear activity. Responses were eliminated by destruction of the vestibular end organs, thus showing that responses depended critically and specifically on the vestibular system. The results demonstrate that the responses are vestibular and the findings provide a scientific basis for using vestibular responses to evaluate vestibular function through ontogeny and senescence in the quail. Accepted: 18 January 1997