Phytoremediation with transgenic trees

In the present paper actual trends in the use of transgenic trees for phytoremediation of contaminated soils are reviewed. In this context a current field trial in which transgenic poplars with enhanced GSH synthesis and hence elevated capacity for phytochelatin production are compared with wildtype plants for the removal of heavy metals at different levels of contamination and under different climatic conditions. The studies are carried out with grey poplar (Populus tremula x P. alba), wildtype plants and plants overexpressing the gene for gamma-glutamylcysteine synthetase (gshI) from E. coli in the cytosol. The expression of this gene in poplar leads to two- to four-fold enhanced GSH concentrations in the leaves. In greenhouse experiments under controlled conditions these transgenic poplars showed a high potential for uptake and detoxification of heavy metals and pesticides. This capacity is evaluated in field experiments. Further aims of the project are to elucidate (a) the stability of the transgene under field conditions and (b) the possibility of horizontal gene transfer to microorganisms in the rhizosphere. The results will help to assess the biosafety risk of the use of transgenic poplar for phytoremediation of soils.     

Evaluation of Transgenic Poplars Over-Expressing Enzymes of Glutathione Synthesis for Phytoremediation of Cadmium

Abstract: Recently, phytoremediation of soils polluted with heavy metals has received a lot of attention. Since glutathione (GSH) and its derivatives (e.g., phytochelatins) play a major role in plant defence against environmental pollutants, we tested the effects of over-expression of bacterial genes for GSH synthesis in poplar on cadmium accumulation. A pilot experiment with CdCl₂ in hydroponics revealed that poplars over-expressing γ-glutamylcysteine synthetase (γ-ECS) accumulated significantly more Cd in root tissue than wild type or glutathione synthetase over-expressing poplars. To test the partitioning of Cd in different organs, poplar lines over-expressing γ-ECS in the cytosol and in chloroplasts were treated with 0.2 mM CdCl₂ in hydroponics. Significant amounts of Cd were translocated to leaves, but significant differences in Cd accumulation were not observed between transgenic and wild type plants. To evaluate these lines for large-scale phytoremediation of cadmium, plants were treated with 2 mM Cd in soil. Over a four-week period, the poplar plants were able to accumulate up to 5.3 mg Cd. Most remarkably, in young leaves of both transgenic lines, Cd was accumulated to concentrations 2.5 - 3 times higher than in the wild type. The increased allocation of cadmium to the young leaves represents a potentional advantage for the phytoremediation process using the same plants over several vegetation periods. The use of transgenic poplar lines with enhanced glutathione production capacity seems to be of particular advantage in highly polluted soils.     

Regulation of glutathione synthesis in leaves of transgenic poplar (Populus tremula X P. alba) overexpressing glutathione synthetase

The poplar hybrid Populus tremula X P. alba was transformed with the Escherichia coli gene for glutathione synthetase ( gsh II ) targetted to the cytosol. Leaves of five lines of transgenic plants exhibited glutathione synthetase activities 15- to 60-fold higher than those of wild-type plants. Total glutathione levels and GSH/GSSG ratios were similar in transgenic and wild-type plants. Precursor feeding experiments with cysteine and γ-glutamylcysteine suggest that glutathione synthesis in the cytoplasm is controlled by a multistep procedure that includes (i) the availability of cysteine, (ii) the availability of γ-glutamylcysteine, and (iii) regulation of the activities of both γ-glutamylcysteine synthetase and glutathione synthetase. However step (ii) may set an upper limit for the cellular glutathione content.     

Over-expression of gsh1 in the cytosol affects the photosynthetic apparatus and improves the performance of transgenic poplars on heavy metal-contaminated soil

Recent studies of transgenic poplars over‐expressing the genes gsh1 and gsh2 encoding γ‐glutamylcysteine synthetase (γ‐ECS) and glutathione synthetase, respectively, provided detailed information on regulation of GSH synthesis, enzymes activities and mRNA expression. In this experiment, we studied quantitative parameters of leaves, assimilating tissues, cells and chloroplasts, mesophyll resistance for CO₂ diffusion, chlorophyll and carbohydrate content in wild‐type poplar and transgenic plants over‐expressing gsh1 in the cytosol after 3 years of growth in relatively clean (control) or heavy metal‐contaminated soil in the field. Over‐expression of gsh1 in the cytosol led to a twofold increase of intrafoliar GSH concentration and influenced the photosynthetic apparatus at different levels of organisation, i.e., leaves, photosynthetic cells and chloroplasts. At the control site, transgenic poplars had a twofold smaller total leaf area per plant and a 1.6‐fold leaf area per leaf compared to wild‐type controls. Annual aboveground biomass gain was reduced by 50% in the transgenic plants. The reduction of leaf area of the transformants was accompanied by a significant decline in total cell number per leaf, indicating suppression of cell division. Over‐expression of γ‐ECS in the cytosol also caused changes in mesophyll structure, i.e., a 20% decrease in cell and chloroplast number per leaf area, but also an enhanced volume share of chloroplasts and intercellular airspaces in the leaves. Transgenic and wild poplars did not exhibit differences in chlorophyll and carotenoid content of leaves, but transformants had 1.3‐fold fewer soluble carbohydrates. Cultivation on contaminated soil caused a reduction of palisade cell volume and chloroplast number, both per cell and leaf area, in wild‐type plants but not in transformants. Biomass accumulation of wild‐type poplars decreased in contaminated soil by more than 30‐fold, whereas transformants showed a twofold decrease compared to the control site. Thus, poplars over‐expressing γ‐ECS in the cytosol were more tolerant to heavy metal stress under field conditions than wild‐type plants according to the parameters analysed. Correlation analysis revealed strong dependence of cell number per leaf area unit, chloroplast parameters and mesophyll resistance with the GSH level in poplar leaves.     

Cell-specific measurement of cytosolic glutathione in poplar leaves

The level of glutathione (GSH) in plants is important in defence reactions against biotic and abiotic stresses and can place considerable demand of the sulphur assimilation pathway. Enzymes involved in sulphur assimilation and GSH metabolism are not evenly distributed between different subcellular compartments or between different cell types in leaves or roots; however, there is little information on the effect that such asymmetries have on the actual GSH concentration in each compartment or cell type. In the present study in situ labelling with monochlorobimane (MCB) in combination with confocal laser scanning microscopy was used to quantify GSH in each of the main cell types of poplar leaves from fluorescence of the GSB conjugate formed. Comparison of results from the in situ approach with total GSH levels measured in vitro by high-performance liquid chromatography suggested that only the cytosolic GSH pool was labelled using this approach. This suggests that an appropriate GST was not present within the chloroplasts to catalyse the conjugation reaction and that chloroplastic GSH does not rapidly exchange with the cytoplasmic pool under the conditions of the assay. Cytosolic GSH levels were between 0.2 and 0.3 mm for both photosynthetic and non-photosynthetic (epidermal) cell types in wild-type poplar leaves. Cytosolic levels increased by around two-fold in transgenic poplars over-expressing bacterial gamma-glutamylsynthetase (gamma-ECS) in the cytosol of all cell types, but there was no concomitant increase in the chloroplastic GSH pool.     

Development of transgenic tobacco plants overexpressing maize glutathione S-transferase I for chloroacetanilide herbicides phytoremediation

Glutathione S-transferases (GSTs, EC 2.5.1.18) are a multigene family of detoxification enzymes that biotransform a wide variety of endogenous and exogenous electrophilic substrates, including herbicides. The isozyme GST I from maize exhibits significant catalytic activity for the chloroacetanilide herbicide alachlor and appears to be involved in its detoxifying process. To establish the in planta ability of GST I to detoxify from alachlor, transgenesis studies were carried out. The gene gstI-6His, which encodes for 6His-tagged GST I, was used for the construction of a binary vector suitable for genetic engineering of tobacco plants (Nicotiana tabacum). Through biolistic method transgenic tobacco plants were obtained. Integration of gstI-6His gene in transgenic tobacco plants genome was confirmed by polymerase chain reaction and Southern blot hybridization. The expression of active GST I was established by Western blot analysis, using anti-6His antibody, and by direct purification of 6-His tagged GST I on Ni-NTA agarose. Primary transformed plants harboring the gstI-6His gene were transferred to MS medium supplemented with alachlor and their phenotype was evaluated. The transgenic plants showed substantially higher tolerance to alachlor compared to non-transgenic plants in terms of root, leaves and vigorous development. These transgenic plants are potentially useful biotechnological tools for the development of phytoremediation system for the degradation of herbicide pollutants in agricultural fields.     

转基因杨树林下种植转基因棉花对转基因杨棉复合系统内节肢动物群落多样性的影响

【目的】转Bt基因棉花和转Bt基因杨树在我国已推广使用。本研究的目的是调查和分析不同转基因杨棉复合系统内地上节肢动物群落多样性变化,为转基因杨树大规模应用提供生态安全方面的数据。【方法】2019年4-10月,在河北任丘采取欧洲黑杨Populus nigra林下种植转基因棉花Gossypium hirsutum的模式,设置转基因杨树 转基因棉花(复合生态系统1)和非转基因杨树-转基因棉花(复合生态系统2)两种杨棉复合系统,调查杨树和棉花地上部植株节肢动物种类和个体数量,比较节肢动物各功能群的物种数量和个体数量以及香农指数、优势集中性指数、均匀度指数等群落多样性指数,并测定了转基因杨树和棉花叶片Bt蛋白含量。【结果】复合生态系统1中转基因杨树和棉花各自植株上鳞翅目种群累计个体数均显著低于复合生态系统2相应植株上的个体数量。转基因杨树上叶甲类累计个体数量显著高于非转基因杨树上的,寄生蜂类累计个体数量则显著低于非转基因杨树上的。在6月5日、6月21日和9月7日的调查中,转基因杨树上的节肢动物群落香农指数显著高于非转基因杨树上的。除鳞翅目害虫外,2个复合系统中棉株上节肢动物各功能群累计个体数量无显著性差异,香农指数、优势集中性指数和均匀度指数随时间变化的趋势基本一致且同一调查时间无显著性差异,仅在8月17调查中复合生态系统1中棉株上节肢动物香农指数显著高于复合生态系统2棉株上的。将杨树和棉花上节肢动物各类群累计数量相加作为系统整体,复合生态系统1和复合生态系统2在香农指数、优势集中性指数和均匀度指数数值上无显著差异。在调查后期,2个复合系统中节肢动物种群均向杨树叶片聚集。转基因棉花叶片Bt蛋白含量在各调查期均显著高于转基因杨树叶片的。【结论】转基因杨树对靶标害虫种群数量有较好的控制作用,对同系统内棉花上鳞翅目害虫数量亦有协同控制作用。转基因杨棉复合系统中地上节肢动物群落结构稳定,对系统内棉花地上部节肢动物群落多样性无显著影响。靶标害虫对转Bt基因杨树的抗性发展需持续监测。     

Chloroplast parameters differ in wild type and transgenic poplars overexpressing gsh1 in the cytosol

Poplar mutants overexpressing the bacterial genes gsh1 or gsh2 encoding the enzymes of glutathione biosynthesis are among the best‐characterised transgenic plants. However, this characterisation originates exclusively from laboratory studies, and the performance of these mutants under field conditions is largely unknown. Here, we report a field experiment in which the wild‐type poplar hybrid Populus tremula × P. alba and a transgenic line overexpressing the bacterial gene gsh1 encoding γ‐glutamylcysteine synthetase in the cytosol were grown for 3 years at a relatively clean (control) field site and a field site contaminated with heavy metals. Aboveground biomass accumulation was slightly smaller in transgenic compared to wild‐type plants; soil contamination significantly decreased biomass accumulation in both wild‐type and transgenic plants by more than 40%. Chloroplasts parameters, i.e., maximal diameter, projection area and perimeter, surface area and volume, surface/volume ratio and a two‐dimensional form coefficient, were found to depend on plant type, leaf tissue and soil contamination. The greatest differences between wild and transgenic poplars were observed at the control site. Under these conditions, chloroplast sizes in palisade tissue of transgenic poplar significantly exceeded those of the wild type. In contrast to the wild type, palisade chloroplast volume exceeded that of spongy chloroplasts in transgenic poplars at both field sites. Chlorophyll content per chloroplast was the same in wild and transgenic poplars. Apparently, the increase in chloroplast volume was not connected to changes in the photosynthetic centres. Chloroplasts of transgenic poplar at the control site were more elongated in palisade cells and close to spherical in spongy mesophyll chloroplasts. At the contaminated site, palisade and spongy cell chloroplasts of leaves from transgenic trees and the wild type were the same shape. Transgenic poplars also had a smaller chloroplast surface/volume ratio, both at the control and the contaminated site. Chloroplast number per cell did not differ between wild and transgenic poplars at the control site. Soil contamination led to suppression of chloroplast replication in wild‐type plants. From these results, we assume that overexpressing the bacterial gsh1 gene in the cytosol interacts with processes in the chloroplast and that sequestration of heavy metal phytochelatin complexes into the vacuole may partially counteract this interaction in plants grown at heavy metal‐contaminated field sites. Further experiments are required to test these assumptions.     

Responses of transgenic poplar (Populus tremula x P. alba) overexpressing glutathione synthetase or glutathione reductase to acute ozone stress: visible injury and leaf gas exchange

Untransformed hybrid poplar (Populus tremula x P. alba) and transgenic lines overexpressing glutathione synthetase (GshS) in the cytosol (200-300-fold) or glutathione reductase (GR) either in the cytosol 5-fold) or in the chloroplast (150-200-fold) were exposed to 0 (control), 100, 200 or 300 nl l^-1 ozone for 3 d for 7 h d^-1. Following acute ozone stress treatments, wild-type and transgenic poplar suffered from visible foliar injury consisting of dark brown necrotic lesions on the laminae. Necrotic lesions were sharply separated from photosynthetically active cells by a band of red-violet discoloured cell lines showing yellow autofluorescence by blue light, and blue autofluorescence by UV-light excitation. When plants were exposed to 100 nl l^-1 ozone, leaf injury was in general negligible, but when 200 and 300 nl l^-1 ozone was applied, in both untransformed poplar and transgenic lines overexpressing GshS or GR up to 60% and 80%, respectively, visible injury developed on mature leaves. The mean percentage of injured leaf area amounted to 20-30% (200 nl l^-1) and 40-60% (300 nl l^-1). Irrespective of transformation, young leaves of poplar trees were only slightly affected by ozone treatments. In support of these observations, net CO2 assimilation rates of mature leaves were decreased by up to 65% (300 nl l^-1 ozone) in wild-type and transformed poplar, whereas net photosynthesis of young leaves remained unaffected even under severe stress conditions. Leaf conductance was significantly decreased by all ozone treatments, but was in the same range in young and mature leaves, and in wild-type and transformed poplar, pre- and post-exposure to ozone. It can therefore be assumed that the ozone doses effectively taken up into the leaf tissue were not dependent on leaf development and that the strength of the ozone stress exerted was similar in all types of poplar trees investigated in this study.From these data it is concluded that: (i) elevated foliar activities of glutathione synthetase or glutathione reductase alone are not sufficient to improve tolerance of hybrid poplar to acute ozone stress, and (ii) the sensitivity of poplar leaves to acute ozone stress is controlled by unknown factors closely related to leaf development rather than by foliar activities of glutathione synthetase and glutathione reductase, or leaf conductance.     

Responses to cadmium in leaves of transformed poplars overexpressing Γ-glutamylcysteine synthetase

Poplars overexpressing a bacterial Γ -glutamylcysteine synthetase ( Γ -ECS) in the cytosol (lines ggs11 and ggs28) had a 30-fold increase in foliar Γ -ECS activity relative to untransformed controls. Foliar Γ -glutamylcysteine ( Γ -EC) was increased by 10-fold while foliar glutathione accumulation increased by up to 3.5-fold in the leaves of the transformants. Untransformed and transformed poplars were grown with different soil concentrations of cadmium (0–1100 μg g⁻¹ soil) for 2 weeks. Cadmium accumulated in the leaves of both transformed and untransformed poplars and growth was inhibited. Growth inhibition and foliar cadmium accumulation were greatest at the highest soil cadmium concentrations in all lines. Exposure to cadmium enhanced the foliar cysteine, Γ -EC and glutathione pools in all lines but less glutathione was present in the leaves of the untransformed controls than the transformants under all growth conditions. Cadmium-induced changes in the activities of malic enzyme, isocitrate dehydrogenase and guaiacol peroxidase were less pronounced in the leaves of the transformed poplars overexpressing Γ -ECS than in the untransformed controls. Glutamate dehydrogenase and glutathione reductase activities were unchanged by exposure to cadmium. We conclude that overexpression of Γ -ECS activity and foliar glutathione accumulation in transformed poplar allows greater tissue cadmium accumulation but has only a marginal effect on cadmium tolerance in poplar.     

Regulation of sulphate assimilation by glutathione in poplars (Populus tremula x P. alba) of wild type and overexpressing gamma-glutamylcysteine synthetase in the cytosol

Glutathione (GSH) is the major low molecular weight thiol in plants with different functions in stress defence and the transport and storage of sulphur. Its synthesis is dependent on the supply of its constituent amino acids cysteine, glutamate, and glycine. GSH is a feedback inhibitor of the sulphate assimilation pathway, the primary source of cysteine synthesis. Sulphate assimilation has been analysed in transgenic poplars (Populus tremula x P. alba) overexpressing gamma-glutamylcysteine synthetase, the key enzyme of GSH synthesis, and the results compared with the effects of exogenously added GSH. Although foliar GSH levels were 3-4-fold increased in the transgenic plants, the activities of enzymes of sulphate assimilation, namely ATP sulphurylase, adenosine 5'-phosphosulphate reductase (APR), sulphite reductase, serine acetyltransferase, and O-acetylserine (thiol)lyase were not affected in three transgenic lines compared with the wild type. Also the mRNA levels of these enzymes were not altered by the increased GSH levels. By contrast, an increase in GSH content due to exogenously supplied GSH resulted in a strong reduction in APR activity and mRNA accumulation. This feedback regulation was reverted by simultaneous addition of O-acetylserine (OAS). However, OAS measurements revealed that OAS cannot be the only signal responsible for the lack of feedback regulation of APR by GSH in the transgenic poplars.     

Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants

Over‐expression of glutamine synthetase (GS, EC 6.3.1.2), a key enzyme in nitrogen assimilation, may be a reasonable approach to enhance plant nitrogen use efficiency. In this work phenotypic and biochemical characterizations of young transgenic poplars showing ectopic expression of a pine cytosolic GS transgene in photosynthetic tissue (Gallardo et al., Planta 210, 19–26, 1999) are presented. Analysis of 22 independent transgenic lines in a 6 month greenhouse study indicated that expression of the pine GS transgene affects early vegetative growth and leaf morphology. In comparison with non‐transgenic controls, transgenic trees exhibited significantly greater numbers of nodes and leaves (12%), and higher average leaf length and width resulting in an increase in leaf area (25%). Leaf shape was not altered. Transgenic poplars also exhibited increased GS activity (66%), chlorophyll content (33%) and protein content (21%). Plant height was correlated with GS content in young leaves, suggesting that GS can be considered a marker for vegetative growth. Molecular and kinetic characterization of GS isoforms in leaves indicated that poplar GS isoforms are similar to their counterparts in herbaceous plants. A new GS isoenzyme that displayed molecular and kinetic characteristics corresponding to the octomeric pine cytosolic GS1 was identified in the photosynthetic tissues of transgenic poplar leaves. These results indicate that enhanced growth and alterations in biochemistry during early growth are the consequence of transgene expression and assembly of pine GS1 subunits into a new functional holoenzyme in the cytosol of photosynthetic cells.     

Study on the biochemical characterization of herbicide detoxification enzyme, glutathione S-transferase

To gain further insight into herbicide detoxification, we studied the herbicide activity and specificity toward glutathione S-transferases from human and rice. In this study, the genes of the plant specific phi and tau class GST enzymes from Oryza sativa (OsGST) and human pi class GST enzyme (hGSTP1-1) were cloned and expressed in Escherichia coli with the pET and pKK vector systems, respectively. The gene products were purified to homogeneity by GSH Sepharose affinity column chromatography. The herbicide specificity of the enzymes was investigated by enzyme-catalyzed conjugation of GSH with chloroacetanilide, diphenylether and chloro-s-triazine herbicides. The hGSTP1-1 showed very high specific activity toward atrazine. On the other hand, the phi class OsGST enzymes showed high specific activity toward chloroacetanilide herbicides, acetochlor, alachlor and metolachlor. The tau class GST enzymes displayed remarkable activity toward the diphenylether herbicide, fluorodifen. From these results, we conclude that the phi and the tau class GST enzymes show herbicide specificities and also they play an important role in the detoxification reaction of plant toward herbicides.     

The expression of a maize glutathione S-transferase gene in transgenic wheat confers herbicide tolerance,both in planta and in vitro

Maize (Zea mays), in common with a number of other important crop species, has several glutathione S-transferase (GST) isoforms that have been implicated in the detoxification of xenobiotics via glutathione conjugation. A cDNA encoding the maize GST subunit GST-27, under the control of a strong constitutive promoter, was introduced into explants of the wheat (Triticum aestivum L.) lines cv. Florida and L88-31 via particle bombardment, using the phosphinothricin acetyltransferase (pat) gene as a selectable marker. All six independent transgenic wheat lines recovered expressed the GST-27 gene. T₁ progeny of these wheat lines were germinated on solid medium containing the chloroacetanilide herbicide alachlor, and tolerance to this herbicide was correlated with GST-27 expression levels. In glasshouse sprays, homozygous T₂ plants were resistant not only to alachlor but also to the chloroacetanilide herbicide dimethenamid and the thiocarbamate herbicide EPTC. These additional GST-27 activities, demonstrated via over-expression in a heterologous host, have not been described previously. T₂ plants showed no enhanced tolerance to the herbicides atrazine (an s-triazine) or oxyfluorfen (a diphenyl ether). In further experiments, T₂ wheat plants were recovered from immature transgenic scutella cultured on medium containing 100 mg/l alachlor, a concentration which killed null segregant and wild-type scutella. These data indicate the potential of the maize GST-27 gene as a selectable marker in wheat transformation.     

Regulation of sulfur nutrition in wild-type and transgenic poplar over-expressing gamma-glutamylcysteine synthetase in the cytosol as affected by atmospheric H2S

This study with poplar (Populus tremula x Populus alba) cuttings was aimed to test the hypothesis that sulfate uptake is regulated by demand-driven control and that this regulation is mediated by phloem-transported glutathione as a shoot-to-root signal. Therefore, sulfur nutrition was investigated at (a) enhanced sulfate demand in transgenic poplar over-expressing gamma-glutamylcysteine (gamma-EC) synthetase in the cytosol and (b) reduced sulfate demand during short-term exposure to H2S. H(2)S taken up by the leaves increased cysteine, gamma-EC, and glutathione concentrations in leaves, xylem sap, phloem exudate, and roots, both in wild-type and transgenic poplar. The observed reduced xylem loading of sulfate after H2S exposure of wild-type poplar could well be explained by a higher glutathione concentration in the phloem. In transgenic poplar increased concentrations of glutathione and gamma-EC were found not only in leaves, xylem sap, and roots but also in phloem exudate irrespective of H(2)S exposure. Despite enhanced phloem allocation of glutathione and its accumulation in the roots, sulfate uptake was strongly enhanced. This finding is contradictory to the hypothesis that glutathione allocated in the phloem reduces sulfate uptake and its transport to the shoot. Correlation analysis provided circumstantial evidence that the sulfate to glutathione ratio in the phloem may control sulfate uptake and loading into the xylem, both when the sulfate demand of the shoot is increased and when it is reduced.     

Disturbance in the allocation of carbohydrates to regenerative organs in transgenic Nicotiana tabacum L

Transgenic tobacco (Nicotiana tabacum L, cv. SR-1) expressing mannitol 1-phosphate dehydrogenase, MTLD, in chloroplasts and myo-inositol O-methyltransferase, IMT1, in the cytosol after crossing of lines which expressed these foreign genes separately has been analysed. Plants expressing both enzymes accumulated mannitol and D-ononitol in amounts comparable to those following single gene transfer and showed phenotypically normal growth during the vegetative stage. Induction of flowering for transgenovar and wild-type occurred at the same time, but during flowering the phenotype of the transformed plants changed. Compared to wild-type, transgenic plants were characterized by curled, smaller upper leaves and elongated stems during flowering; incomplete development of flower buds with shorter sepals and pedicels resulted in increased abortion. Flowers completing development were normal. The vegetative biomass of the transformed plants was slightly higher than that of wild-type. Concentrations of soluble sugars and potassium were lower than in wild-type only in the apical parts of the transgenic plants. Both enzymes, under control of the CaMV 35S promoter, promoted accumulation of mannitol and D-ononitol in the youngest leaves close to the vegetative meristem and in flowers, suggesting that their presence could signal lower sink demand leading to a decrease in carbon import to flowers and developing seed capsules. The interpretation here is that increases of inert carbohydrates in developing sinks interfere with metabolism, such as respiration or glycolysis. This interference may be less significant in source tissues during vegetative growth than in sink tissues during seed development.     

Photoinhibition of photosystem II in tobacco plants overexpressing glutathione reductase and poplars overexpressing superoxide dismutase

We studied photoinhibition in two cultivars of tobacco ( Nicotiana tabacum L.) expressing the bacterial gor gene in the cytosol and in four lines of poplar ( Populus tremula × P. alba ) expressing the FeSOD gene of Arabidopsis thaliana in the chloroplast. The respective total activities of glutathione reductase (EC 1.6.4.2) in leaves of gor tobaccos and superoxide dismutase (EC 1.15.1.1) in the FeSOD poplars were 5–8 times higher than in the respective untransformed control plants. Leaves of control and transformed plants were subjected to high-light stress at 20°C, and photoinhibition of photosystem II (PSII) was measured by oxygen evolution and chlorophyll fluorescence. The leaves were illuminated both in the presence and absence of lincomycin, which inhibits chloroplast protein synthesis. In both cases, the time course of loss of PSII activity was identical in plants overproducing superoxide dismutase (SOD) and in the untransformed controls, suggesting that the ability to convert superoxide to hydrogen peroxide is not a limiting factor in protection against photoinhibition, or in the repair of photoinhibitory damage or that the site of O₂⁻ production is not accessible to the transgene product. The rate constant of photoinhibition, measured in lincomycin-treated leaves, was smaller in glutathione reductase (GR) overproducing tobacco cv. Samsun than in the respective wild-type, but this difference was not seen in cv. Bel W3. The steady-state level of PSII activity measured when the PSII repair cycle was allowed to equilibrate with photoinhibitory damage under high light was not higher in the GR overproducing cv. Samsun, suggesting that the repair of photoinhibitory damage was not enhanced in plants overproducing GR in the cytosol.     

Overexpression of poplar cellulase accelerates growth and disturbs the closing movements of leaves in sengon

In this study, poplar (Populus alba) cellulase (PaPopCel1) was overexpressed in a tropical Leguminosae tree, sengon (Paraserianthes falcataria), by the Agrobacterium tumefaciens method. PaPopCel1 overexpression increased the length and width of stems with larger leaves, which showed a moderately higher density of green color than leaves of the wild type. The pairs of leaves on the transgenic plants closed more slowly during sunset than those on the wild-type plants. When main veins from each genotype were excised and placed on a paper towel, however, the leaves of the transgenic plants closed more rapidly than those of the wild-type plant. Based on carbohydrate analyses of cell walls, the leaves of the transgenic plants contained less wall-bound xyloglucan than those of the wild-type plants. In situ xyloglucan endotransglucosylase activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, occurred in the parenchyma cells (motor cells) of the petiolule pulvinus attached to the main vein, although the transgenic plant incorporated less whole xyloglucan than the wild-type plant. These observations support the hypothesis that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase, which loosens xyloglucan intercalation, resulting in an irreversible wall modification. This process could be the reason why the overexpression of poplar cellulase both promotes plant growth and disturbs the biological clock of the plant by altering the closing movements of the leaves of the plant.