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 regulation of component processes of photosynthesis in transgenic tobacco with decreased phosphoribulokinase activity

Tobacco plants (Nicotiana tabacum L.) transformed with an inverted cDNA encoding ribulose 5-phosphate kinase (phosphoribulokinase,PRK; EC 2.7.1.19) were employed to study the in vivo relationship between photosynthetic electron transport and the partitioning of electron transport products to major carbon metabolism sinks under conditions of elevated ATP concentrations and limited ribulose 1,5-bisphosphate (RuBP) regeneration. Simultaneous measurements of room temperature chlorophyll fluorescence and CO₂ gas exchange were conducted on intact leaves. Under ambient CO₂ concentrations and light intensities above those at which the plants were grown, transformants with only 5% of PRK activity showed down-regulation of PS II activity and electron transport in response to a decrease in net carbon assimilation when compared to wild-type. This was manifested as a decline in the efficiency of PS II electron transport (_{PS II}), an increase in dissipation of excess absorbed light in the antennae of PS II and a decline in: total linear electron transport (J₁), electron transport dedicated to carbon assimilation (J_A) and electron transport allocated to photorespiration (J_L). The transformants showed no alteration in the Rubisco specificity factor measured in vitro and calculated in vivo but had a relatively smaller ratio of RuBP oxygenation to carboxylation rates (v_o/v_c), due to a higher CO₂ concentration at the carboxylation site (C_c). The relationship between _{PS II} and _{CO 2}was similar in transformants and wild-type under photorespiratory conditions demonstrating no change in the intrinsic relationship between PS II function and carbon assimilation, however, a novel result of this study is that this similar relationship occurred at different values of quantum flux, J₁, J_A, J_L and v_o/v_c in the transformant. For both wild-type and transformants, an assessment was made of the possible presence of a third major sink for electron transport products, beside RuBP oxygenation and carboxylation, the data provided no evidence for such a sink.Abbreviations C_c CO₂ concentration at the site of carboxylation - C_i intercellular CO₂ concentration - g_m mesophyll conductance to CO₂ - J₁ total linear electron flow - J_A linear electron flow allocated to CO₂ assimilation - J_c linear electron flow supporting carbon reduction and oxidation cycles - J_L linear electron flow allocated to photorespiration (RuBP oxygenation and fixation of released photorespiratory CO₂) - PRK phosphoribulokinase - q_P, q_N coefficients for photochemical and non-photochemical quenching of fluorescence respectively - Rubisco ribulose 1,5-bisphosphate carboxylase-oxygenase - S Rubisco specificity to CO₂/O₂ - v_c, v_o rates of RuBP carboxylation and RuBP oxygenation, respectively - _{CO 2} relative quantum yield of CO₂ assimilation - _C maximum _{CO 2} under non-photorespiratory conditions - _exc the efficiency of excitation capture by open PS II centres - _{PS II} relative quantum yield of PS II electron transport     

Reduction in phosphoribulokinase activity by antisense RNA in transgenic tobacco: effect on CO2 assimilation and growth in low irradiance

To quantify the importance of the Calvin cycle enzyme phosphoribulokinase (PRK) in photosynthesis and to perturb photosynthesis without large direct reductions in leaf protein content, tobacco plants (Nicotiana tabacum L.) were transformed with an inverted cDNA encoding tobacco PRK. A population of plants expressing antisense RNA and a range of PRK activities from wild-type to less than 5% of wild-type were obtained. CO₂ assimilation under the growing conditions (330 µmol photons m^?2 sec^?1, 350 µbar CO₂, 25°C) was not inhibited until more than 85% of PRK activity had been removed. With reduction in PRK activity of between 85 and 95%, assimilation rates and amounts of chlorophyll compared with wild-type were reduced by up to half. Decreased absorption of light by leaves with less chlorophyll accounte0d for only a small part of the reduction in assimilation rate. When PRK activity was below 15% of wild-type, amounts of ribulose-5-phosphate, ribose-5-phosphate, ATP and fructose-6-phosphate were 1.5- to fivefold higher and levels of ribulose-1,5-bisphosphate, 3-phosphoglyceric acid and ADP 1.5- to fourfold lower than in wild-type. It is estimated that these changes maintained flux through PRK to realise the assimilation rates observed. A possible shift of control within the Calvin cycle towards fructose-1,6-bisphosphatase in plants with low PRK is discussed. Amounts of hexoses and starch in particular were reduced in plants expressing the lowest PRK activities; amounts of sucrose were little affected. Lower CO₂ assimilation in plants with low PRK activity correlated with reduced relative growth rate of shoots and delayed flowering, but there was no effect on specific leaf area. It is concluded that (i) in wild-type plants grown in constant low light, PRK has a flux-control coefficient for CO₂ assimilation of zero, and that even when amounts of PRK are reduced 20-fold relative to wild-type, altered amounts of metabolites compensate for much of the reduction in PRK protein; (ii) in plants where there is a 95% reduction in amounts of PRK, photosynthesis was reduced twofold without large changes in leaf protein content or leaf geometry.     

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     

Reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase content by antisense RNA reduces photosynthesis in transgenic tobacco plants

A complementary DNA for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was cloned from tobacco (Nicotiana tabacum) and fused in the antisense orientation to the cauliflower mosaic virus 35S promoter. This antisense gene was introduced into the tobacco genome, and the resulting transgenic plants were analyzed to assess the effect of the antisense RNA on Rubisco activity and photosynthesis. The mean content of extractable Rubisco activity from the leaves of 10 antisense plants was 18% of the mean level of activity of control plants. The soluble protein content of the leaves of anti-small subunit plants was reduced by the amount equivalent to the reduction in Rubisco. There was little change in phosphoribulokinase activity, electron transport, and chlorophyll content, indicating that the loss of Rubisco did not affect these other components of photosynthesis. However, there was a significant reduction in carbonic anhydrase activity. The rate of CO₂ assimilation measured at 1000 micromoles quanta per square meter per second, 350 microbars CO₂, and 25°C was reduced by 63% (mean value) in the antisense plants and was limited by Rubisco activity over a wide range of intercellular CO₂ partial pressures (p_i). In control leaves, Rubisco activity only limited the rate of CO₂ assimilation below a p_i of 400 microbars. Despite the decrease in photosynthesis, there was no reduction in stomatal conductance in the antisense plants, and the stomata still responded to changes in p_i. The unchanged conductance and lower CO₂ assimilation resulted in a higher p_i, which was reflected in greater carbon isotope discrimination in the leaves of the antisense plants. These results suggest that stomatal function is independent of total leaf Rubisco activity.     

Modification of carbonic anhydrase activity by antisense and over-expression constructs in transgenic tobacco

The activity and location of carbonic anhydrase has been modified by transformation of tobacco with antisense and over-expression constructs. Antisense expression resulted in the inhibition of up to 99% of carbonic anhydrase activity but had no significant impact on net CO₂ assimilation. Stomatal conductance and susceptibility to water stress appeared to increase in response to the decline in carbonic anhydrase activity. An over-expression construct designed to increase cytosolic carbonic anhydrase abundance resulted in a significant increase in net activity, a small increase in stomatal conductance but little impact on CO₂ assimilation. Chloroplastic carbonic anhydrase activity was enhanced by the expression of an additional construct which targeted the polypeptide to the organelle. The increase in chloroplastic carbonic anhydrase appeared to be accompanied by a concomitant increase in Rubisco activity.     

Cadmium toxicity affects photosynthesis and plant growth at different levels

In this article we discuss and update some of the effects of Cd toxicity on the photosynthetic apparatus in a model crop Lactuca sativa. Seeds of L. sativa were germinated in solutions with 0, 1, 10 and 50 μM of Cd(NO₃)₂ and then transferred to a hydroponic culture medium. After 28 days, the effects of Cd on the photosynthetic apparatus of lettuce were analysed. Exposure of lettuce to 1 μM Cd(NO₃)₂ affected already plant growth (dry biomass), but, did not induce serious damages in the photosynthetic apparatus. However, increasing concentrations of this metal to 10 and 50 μM promoted a strong reduction of the maximum photochemical efficiency of PSII and an impairment of net CO₂ assimilation rate, putatively due to Rubisco activity decrease. This ultimately results in a strong inhibition of plant growth. Nutrient uptake and carbohydrate assimilation were also severely affected by Cd.     

The impact of decreased Rubisco on photosynthesis, growth, allocation and storage in tobacco plants which have been transformed with antisense rbcS

Transgenic tobacco plants tranformed with antisense to rbcS to decrease expression of ribulose-1,5–bisphosphate carboxylase-oxygenase (Rubisco) have been used to investigate (a) whether Rubisco is limiting for photosynthesis and plant growth and (b) whether biomass allocation and storage of carbohydrate and nitrogen are regulated in response to decreased rate of photosynthesis. The rate of photosynthesis (measured in growth conditions) and plant growth were not strongly inhibited until almost half of the Rubisco was removed. When Rubisco was decreased further there was a large decrease of photosynthesis and plant growth. When photosynthesis decreased in the ‘antisense’ plants there was an increase in the shoot/root ratio and the specific leaf area. As a result, the leaf area ratio (leaf area per g plant dry weight) increased 3–4–fold. This shows that tobacco compensates for decreased photosynthesis by maximizing leaf area. The decrease of photosynthesis also resulted in lower starch and free hexose in the leaf, but the volume of the diurnal starch turnover was largely maintained. This indicates that partitioning to starch is regulated to decrease non-productive accumulation of starch, but still maintain a pool of transient starch for export during the night. The decrease of photosynthesis was also accompanied by a large increase of the nitrogen/ carbon balance, due to a large accumulation of nitrate in the leaf. This shows that assimilation of nitrate is inhibited in response to low availability of photo-synthate.     

Reduction of ribulose biphosphate carboxylase activase levels in tobacco (Nicotiana tabacum) by antisense RNA reduces ribulose biphosphate carboxylase carbamylation and impairs photosynthesis.

The in vivo activity of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is modulated in response to light intensity by carbamylation of the active site and by the binding of sugar phosphate inhibitors such as 2'-carboxyarabinitol-1-phosphate (CA 1P). These changes are influenced by the regulatory protein Rubisco activase, which facilitates the release of sugar phosphates from Rubisco's catalytic site. Activase levels in Nicotiana tabacum were reduced by transformation with an antisense gene directed against the mRNA for Rubisco activase. Activase-deficient plants were photosynthetically impaired, and their Rubisco carbamylation levels declined upon illumination. Such plants needed high CO2 concentrations to sustain reasonable growth rates, but the level of carbamylation was not increased by high CO2. The antisense plants had, on average, approximately twice as much Rubisco as the control plants. The maximum catalytic turnover rate (k cat) of Rubisco decreases in darkened tobacco leaves because of the binding of CA 1P. The dark-to-light increase in k cat that accompanies CA 1P release occurred to similar extents in antisense and control plants, indicating that normal levels of activase were not essential for CA 1P release from Rubisco in the antisense plants. However, CA 1P was released in the antisense plants at less than one-quarter of the rate that it was released in the control plants, indicating a role for activase in accelerating the release of CA 1P.     

Intercropping effect on root growth and nitrogen uptake at different nitrogen levels

Aims Intercropping legumes and non-legumes may affect the root growth of both components in the mixture, and the non-legume is known to be strongly favored by increasing nitrogen (N) supply. The knowledge of how root systems affect the growth of the individual species is useful for understanding the interactions in intercrops as well as for planning cover cropping strategies. The aim of this work was (i) to determine if different levels of N in the topsoil influence root depth (RD) and intensity of barley and vetch as sole crops or as an intercropped mixture and (ii) to test if the choice of a mixture or the N availability in the topsoil will influence the N uptake by deep roots.Methods In this study, we combined rhizotron studies with root extraction and species identification by microscopy with studies of growth, N uptake and 15 N uptake from deeper soil layers, for studying the root interactions of root growth and N foraging for barley (Hordeum vulgare L.) and vetch (Vicia sativa L.), frequently grown in mixtures as cover crops. N was added at 0 (N0), 50 (N1) and 150 (N2) kg N ha^-1. The roots discrimination relying on the anatomical and morphological differences observed between dicots and monocots proved to be a reliable method providing valuable data for the analysis.Important findings The intercrop and the barley attained slightly higher root intensity (RI) and RD than the vetch, with values around 150 crosses m^-1 and 1.4 m, respectively, compared to 50 crosses m^-1 and 0.9 m for the vetch. At deep soil layers, intercropping showed slightly larger RI values compared to the sole-cropped barley. The barley and the intercropping had larger root length density (RLD) values (200–600 m m ?3) than the vetch (25–130) at 0.8–1.2 m depth. The topsoil N supply did not show a clear effect on the RI, RD or RLD; however, increasing topsoil N favored the proliferation of vetch roots in the intercropping at deep soil layers, with the barley:vetch root ratio ranging from 25 at N0 to 5 at N2. The N uptake of the barley was enhanced in the intercropping at the expense of the vetch (from ~100mg plant^-1 to 200). The intercropped barley roots took up more labeled nitrogen (0.6mg 15 N plant^-1) than the sole-cropped barley roots (0.3mg 15 N plant^-1) from deep layers.     

不同氮水平下瘤突苍耳、苍耳及其杂交种形态、光合及生长特征比较

入侵种与本地种杂交可能会改变其入侵性。为探讨入侵种与本地种杂交是否能促进植物入侵, 我们通过盆栽实验比较了高、中和低3种氮水平下入侵植物瘤突苍耳(Xanthium strumarium)、本地近缘种苍耳(X. sibiricum)及两者杂交种(X. strumarium♀ × X. sibiricum♂)的形态、光合及生长特征的差异。结果表明, 杂交种的总生物量在中氮和高氮水平下显著低于瘤突苍耳而高于苍耳。然而, 杂交种的茎粗在低氮水平下显著高于两个亲本, 叶绿素总含量和蒸腾速率在高氮水平下显著高于瘤突苍耳, 相对生长速率在低氮和高氮水平下显著高于两个亲本。此外, 在3种氮水平下瘤突苍耳的株高均显著小于苍耳, 而杂交种的株高在中氮和高氮水平下均与苍耳无显著差异。这些特性有可能提高了杂交种对资源的捕获和利用能力, 使其不仅能适应贫瘠的养分环境, 还能在有利条件下扩大自身优势。杂交带来较高的生长速率可能与瘤突苍耳的入侵性相关。     

Altered Rubisco activity and amounts of a daytime tightbinding inhibitor in transgenic tobacco expressing limiting amounts of phosphoribulokinase

Transgenic tobacco with RuBP-limited photosynthetic assimilationdue to a 95% reduction in phosphoribulokinase activity, hadhigher specific activities of Rubisco in fresh extracts andafter full activation, than in the wild type. Differences inthe amounts of a daytime tight-binding inhibitor were sufficientto contribute significantly to these activity differences. Key words: Nicotiana tabacum, transgenic plant, phosphoribulokinase, Rubisco, tight-binding inhibitor     

Increased sedoheptulose-1,7-bisphosphatase activity in transgenic tobacco plants stimulates photosynthesis and growth from an early stage in development

Activity of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase) was increased by overexpression of an Arabidopsis (Arabidopsis thaliana) cDNA in tobacco (Nicotiana tabacum) plants. In plants with increased SBPase activity, photosynthetic rates were increased, higher levels of Suc and starch accumulated during the photoperiod, and an increase in leaf area and biomass of up to 30% was also evident. Light saturated photosynthesis increased with increasing SBPase activity and analysis of CO2 response curves revealed that this increase in photosynthesis could be attributed to an increase in ribulose 1,5-bisphosphate regenerative capacity. Seedlings with increased SBPase activity had an increased leaf area at the 4 to 5 leaf stage when compared to wild-type plants, and chlorophyll fluorescence imaging of these young plants revealed a higher photosynthetic capacity at the whole plant level. Measurements of photosynthesis, made under growth conditions integrated over the day, showed that mature plants with increased SBPase activity fixed 6% to 12% more carbon than equivalent wild-type leaves, with the young leaves having the highest rates. In this paper, we have shown that photosynthetic capacity per unit area and plant yield can be increased by overexpressing a single native plant enzyme, SBPase, and that this gives an advantage to the growth of these plants from an early phase of vegetative growth. This work has also shown that it is not necessary to bypass the normal regulatory control of SBPase, exerted by conditions in the stroma, to achieve improvements in carbon fixation.     

Elevated sucrose-phosphate synthase activity in transgenic tobacco sustains photosynthesis in older leaves and alters development

Constitutive over-expression of a maize sucrose-phosphate synthase (SPS) gene in tobacco (Nicotiana tabacum) had major effects on leaf carbohydrate budgets with consequences for whole plant development. Transgenic tobacco plants flowered earlier and had greater flower numbers than wild-type plants. These changes were not linked to modified source leaf carbon assimilation or carbon export, although sucrose to starch ratios were significantly higher in leaves expressing the transgene. The youngest and oldest leaves of plants over-expressing SPS had up to 10-fold wild-type maximal extractable SPS activity, but source leaf SPS activities were only 2-3 times greater in these lines than in the wild type. In the oldest leaves, where the expression of the transgene led to the most marked enhancement in SPS activity, photosynthesis was also increased. It was concluded that these increases in the capacity for sucrose synthesis and carbon assimilation, particularly in older leaves, accelerate the whole plant development and increase the abundance of flowers without substantial changes in the overall shoot biomass.     

Prediction of growth rate at different light levels from measured photosynthesis and respiration rates

Light integrators with a linear response are not suitable for measuring the light climates of plants because plants are not linear integrators. It should be possible to make a quantitative allowance for this nonlinearity by using the CO₂ uptake curve of the plant. To test this, we have subjected white clover plants to different levels of constant light, comparing the rate of increase of total dry matter with the net rate of uptake of CO₂ per day. Temperature, humidity, daylength and nutrient supply were kept constant. The growth rate calculated from CO₂ uptake agreed well with the observed rate over the light levels tested (3.7-88 w·m⁻², 0.4-0.7 micron: 1 w·m⁻² = 10³ erg · sec⁻¹ cm⁻²). All plants put on weight over the few days of the experiment, even those placed at light levels below their compensation point. The plants adapted their respiration rates to be a constant proportion of their growth rates. Most of the adaptation occurred within 24 hours of the light change. The adaptation of respiration has implications for models of light/growth relations in plant communities, almost all of which assume that respiration is proportional to leaf area and independent of growth rate or light level. The only model which does not is that of de Wit, and this gave good agreement with our results.     

Decreased sucrose-6-phosphate phosphatase level in transgenic tobacco inhibits photosynthesis,alters carbohydrate partitioning,and reduces growth

The aim of this work was to examine the role of sucrose-6-phosphate phosphatase (SPP; EC 3.1.3.24) in photosynthetic carbon partitioning. SPP catalyzes the final step in the pathway of sucrose synthesis; however, until now the importance of this enzyme in plants has not been studied by reversed-genetics approaches. With the intention of conducting such a study, transgenic tobacco plants with reduced SPP levels were produced using an RNA interference (RNAi) strategy. Transformants with less than 10% of wild-type SPP activity displayed a range of phenotypes, including those that showed inhibition of photosynthesis, chlorosis, and reduced growth rates. These plants had strongly reduced levels of sucrose and hexoses but contained 3–5 times more starch than the control specimens. The leaves were unable to export transient starch during extended periods of darkness and as consequence showed a starch- and maltose-excess phenotype. This indicates that no alternative mechanism for carbon export was activated. Inhibition of SPP resulted in an approximately 1,000-fold higher accumulation of sucrose-6-phosphate (Suc6P) compared to wild-type leaves, whereas the content of hexose-phosphates was reduced. Although the massive accumulation of Suc6P in the cytosol of transgenic leaves was assumed to impair phosphate-recycling into the chloroplast, no obvious signs of phosphate-limitation of photosynthesis became apparent. 3-Phosphoglycerate (3-PGA) levels dropped slightly and the ATP/ADP ratio was not reduced in the transgenic lines under investigation. It is proposed that in SPP-deficient plants, long-term compensatory responses give rise to the observed acceleration of starch synthesis, increase in total cellular Pi content, decrease in protein content, and related reduction in photosynthetic activity.