Improved rates of CO2-fixation by intact chloroplasts isolated in media with KCl as the osmoticum

Intact chloroplasts were isolated from spinach leaves using media with either 330 mM sorbitol or 200 mM KCl as the osmoticum. Chloroplasts isolated in KCl exhibited higher rates of CO₂-dependent oxygen evolution in nine out of ten experiments, the average increase being 43%. Chloroplasts isolated in KCl routinely achieved rates of CO₂-dependent oxygen evolution of 200–300 mol·mg chlorophyll^-1·hour^-1 at 20°C. Intact chloroplasts were also isolated in media with 200 mM NaCl or choline chloride but the rates of CO₂ fixation were not superior to those isolated in sorbitol media. The K⁺ content of chloroplasts isolated in KCl media was higher than for chloroplasts isolated in sorbitol. It is suggested that the use of KCl as an osmoticum prevents the loss of chloroplast K⁺ which can occur during isolation in sorbitol media. Chloroplasts isolated in KCl lost, on average, 36% of the initial CO₂ fixation activity after storage for four hours on ice, compared to 24% loss of activity for chloroplasts isolated in sorbitol. This increased loss of activity was not observed if KCl was used in the grinding medium and sorbitol or glycinebetaine in the resuspension media. For measurement of the maximum photosynthetic capacity in vitro, the use of KCl in the grinding medium may be better than sorbitol.Abbreviations BSA bovine serum albumin - Chl chlorophyll - Pi inorganic orthophosphate - EDTA ethlenediamine tetraacetic acid     

Osmoregulation and cell composition in salt-adaptation of Nostoc muscorum

Adaptation to salt in the cyanobacterium Nostocmuscorum, is composed of a few mechanisms which together lead to the generation of a salt-tolerant cell. The initial mechanism combines a stimulation of photosynthetic activity with the accumulation of sucrose as an osmoregulator. The secondary mechanism involves the adaptation of N₂ fixation activity and protein biosynthesis. The adaptation is most efficient in response to NaCl-induced stress and functions only partially under stress induced by either KCl or a nonionic osmoticum such as mannitol.     

Assessment of glycinebetaine and proline compartmentation by analysis of isolated beet vacuoles

Vacuoles isolated from storage root tissue of red beet (Beta vulgaris L.) do not leak significant quantities of betanin, sucrose, Na⁺ or K⁺ during isolation. This indicates that analysis of vacuoles in vitro gives meanigful information about the compartmentation of solutes in vivo. Preparations of vacouoles were used to determine the distribution of glycinebetaine and proline between vacuole and cytoplasm in beet cells. Both compounds were detected in preparations of isolated beet vacuoles. In the case of glycinebetaine it was shown that this solute was associated with the vacuoles, not with the small number of other organelles which contaminated the preparations. The vacuolar pool accounted for 26 to 84% of the total tissue glycinebetaine and 17 to 57% of the proline. Concentrations of these compounds in vacuole and cytoplasm were calculated and were always higher in the cytoplasm than in the vacuole. The concentration gradient across the tonoplast varied considerably. The significance of these results is discussed in relation to the hypothesis that glycinebetaine and proline function as benign cytoplasmic osmotica.Abbreviations A₅₃₇ absorbance at 537 nm - MES 2-(N-morpholino)-ethanesulphonic acid - Na₂EDTA ethylenediaminetetraacetic acid, disodium salt - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl)methylamine     

The Effect of Different Salts of Sodium and Potassium on the Accumulation of Glycinebetaine in Atriplex Prostrata

Atriplex prostrata was grown for one month in nutrient solutions with NaCl, KCl, Na₂SO₄, and K₂SO₄ (at osmotic potentials of 0, –0.75, –1.00, and –1.50 MPa). Plants treated with K₂SO₄ had less glycinebetaine at –1.0 and –1.50 MPa than those treated with Na⁺ salts, probably due to the inhibitory effects of K⁺ on glycinebetaine accumulation.     

Enzyme activities in concentrated solutions of glycinebetaine and other solutes

The activities of a number of enzymes in concentrated solutions of glycinebetaine and other solutes have been studied. Glycinebetaine, in contrast to electrolytes such as NaCl, was found to be noninhibitory up to 500 mM. This is compatible with the postulated role of glycinebetaine in cytoplasmic osmoregulation. Partial protection against NaCl inhibition was afforded by glycinebetaine in some cases. More detailed studies on glycinebetaine —NaCl-enzyme interactions were carried out using malate dehydrogenase (decarboxylating) from Hordeum vulgare.Abbreviations TES N-tris[hydroxymethyl]methyl-2-aminoethane sulphonic acid - MES 2[N-Morpholino]ethane sulphonic acid     

Transformation of Synechococcus with a gene for choline oxidase enhances tolerance to salt stress

Choline oxidase, isolated from the soil bacterium Arthrobacter globiformis, converts choline to glycinebetaine (N-trimethylglycine) without a requirement for any cofactors. The gene for this enzyme, designated codA, was cloned and introduced into the cyanobacterium Synechococcus sp. PCC 7942. The codA gene was experssed under the control of a strong constitutive promoter, and the transformed cells accumulated glycinebetaine at intracellular levels of 60–80 mM. Consequently the cells acquired tolerance to salt stress, as evaluated in terms of growth, accumulation of chlorophyll and photosynthetic activity.     

Enhanced germination under high-salt conditions of seeds of transgenicArabidopsis with a bacterial gene (codA) for choline oxidase

Arabidopsis thaliana was transformed previously with thecodA gene from the soil bacteriumArthrobacter globiformis. This gene encodes choline oxidase, the enzyme that converts choline to glycinebetaine. Transformation with thecodA gene significantly enhanced the tolerance of transgenic plants to low temperature and high-salt stress. We report here that seeds of transgenic plants that expressed thecodA gene were also more tolerant to salt stress during germination than seeds of non-transformed wild-type plants. Seedlings of transgenic plants grew more rapidly than those of wild-type plants under salt-stress conditions. Furthermore, exogenously applied glycinebetaine was effective in alleviating the harmful effects of salt stress during germination of seeds and growth of young seedlings, a result that suggests that it was glycinebetaine that had enhanced the tolerance of the transgenic plants. These observations indicate that synthesis of glycinebetaine in transgenic plantsin vivo, as a result of the expression of thecodA gene, might be veryuseful in improving the ability of crop plants to tolerate salt stress. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

Electron microscopic structure and oxygen evolution activity of thylakoids from Avicennia marina prepared under different osmotic and ionic conditions

Abstract Stacking of thylakoid membranes in vitro was assessed using electron microscopy. Grana stacks of spinach thylakoids formed when 5 mol m^?3 MgCl₂ was present, but no stacking of thylakoids from the mangrove Avicennia marina occurred in the presence of 10 mol m^?3? MgCl₂. Isolation of mangrove thylakoids with a high osmotic strength medium did not induce grana formation if the medium consisted only of sorbitol or glycinebetaine. Addition of cations to the high osmotic strength medium did induce some loose-grana formation, with divalent cations being more effective than monovalent cations. Glycinebetaine was a better osmoticum than sorbitol for grana formation provided divalent cations had been added. Oxygen evolution activity of the preparations was influenced by the amount of membrane stacking, with the preparations with the greatest amount of stacked membrane having the highest activity. Isolation with sorbitol or glycinebetaine based media did not alter this pattern, nor did assay in sorbitol or glycinebetaine. Mangrove thylakoids have a requirement for both a high osmotic strength and divalent cations for grana formation in vitro which may be related to the low water potential of the plant environment in vivo.     

Glycinebetaine biosynthesis and its control in detached secondary leaves of spinach

In secondary leaves from spinach plants pretreated in vermiculite for 24 h with 300 mM NaCl, glycinebetaine accumulated at a rate of circa 0.16 mol 100 g^-1 Chl d^-1 (2 mol g^-1 FW d^-1), about three times the rate of control plants. The soluble carbohydrate and free amino acid contents did not increase significantly following salinisation until after 4 d when the relative growth rate also decreased. Leaf proline levels remained very low throughout the experimental period. K⁺ on a tissue water basis remained constant at 200 mM while Cl^- and Na⁺ levels increased linearly to reach 175 and 100 mM respectively after 5 d of saline treatment. The osmotic pressure of leaf tissue also increased from 300 to 500 mosmol kg^-1. These experimental conditions were considered suitable to study glycinebetaine biosynthesis and its induction by salinity in the absence of marked growth inhibition or metabolic disturbance. Radioactive labelled [¹⁴C]serine, ethanolamine and choline (all 1 mol, 13.3 MBq in 10 l) were fed to detached secondary leaves via the petiole 24 h after the exposure of plants to salt. The rate of isotope incorporation into water soluble products, lipids and residue was measured over a further 24 h. The major metabolic fate of exogenous [¹⁴C]choline and [¹⁴C]ethanolamine was incorporation into glycinebetaine while less ¹⁴C-label was found in phosphatidyl choline and phosphatidyl ethanolamine. Incorporation rates were identical in control and salinised leaves and were adequate to account for observed values of glycinebetaine accumulation previously reported in spinach. In contrast the labelling of glycinebetaine from [¹⁴C]serine was twice as great in salinated plants as in the controls. These results, together with short term labelling experiment with [¹⁴C]ethanolamine using leaf slices, were consistent with the formation of glycinebetaine via serine, ethanolamine and its methylated derivatives to choline with some control being exerted at the serine level. However a flux through the phosphorylated intermediates is not excluded.From a consideration of these results and the published data on barley subjected to water stress (Hanson and Scott, 1980 Plant Physiol. 66, 342–348) there appear to be significant differences in the biosynthetic pathways in spinach and barley.Abbreviations BHT butylated hydroxytoluerte (2,6-di-tert-butyl-4-methylphenol) - C₁ one-carbon fragment - 1,2DG diglyceride moiety - DW day weight - MCW methanol-chloroform-water (12:5:1, by vol.) - PA phosphatidic acid - PC phosphatidyl choline - PMME phosphatidyl monomethylethanolamine - PDME phosphatidyl dimethylethanolamine - PE phosphatidyl ethanolamine - PPO 2,5-diphenyloxazole - POPOP 1,4-bis(5-phenyloxazoyl) benzene     

Improved Performance of Transgenic Glycinebetaine-Accumulating Rice Plants under Drought Stress

Plasmid DNA (pChlCOD), containing the selectable hygromycin phosphotransferase hpt gene for hygromycin B resistance and the Arthrobacter globiformis codA gene for choline oxidase which catalyzes the direct conversion of choline to glycinebetaine, was delivered into rice plants using Agrobacterium-mediated gene transfer via scutellum-derived calli. Southern, Northern and Western blot analyses demonstrated that the foreign gene had been transferred, integrated into rice chromosomal DNA and expressed. Drought test indicated that glycinebetaine acts as an osmoprotectant and its production in transgenic rice plant helped the cells to maintain osmotic potential and increased root growth, and thus enhanced the ability of the plants to tolerate water deficit This revised version was published online in July 2006 with corrections to the Cover Date.     

Structural changes in the vacuole and cytoskeleton are key to development of the two cytoplasmic domains supporting single-cell C<Subscript>4</Subscript> photosynthesis in <Emphasis Type="Italic">Bienertia sinuspersici</Emphasis>

Bienertia sinuspersici Akhani has an unusual mechanism of C₄ photosynthesis which occurs within individual chlorenchyma cells. To perform C₄, the mature cells have two cytoplasmic compartments consisting of a central (CCC) and a peripheral (PCC) domain containing dimorphic chloroplasts which are interconnected by cytoplasmic channels. Based on leaf development studies, young chlorenchyma cells have not developed the two cytoplasmic compartments and dimorphic chloroplasts. Fluorescent dyes which are targeted to membranes or to specific organelles were used to follow changes in cell structure and organelle distribution during formation of C₄-type chlorenchyma. Chlorenchyma cell development was divided into four stages: 1—the nucleus and chloroplasts occupy much of the cytoplasmic space and only small vacuoles are formed; 2—development of larger vacuoles, formation of a pre-CCC with some scattered chloroplasts; 3—the vacuole expands, cells have directional growth; 4—mature stage, cells have become elongated, with a distinctive CCC and PCC joined by interconnecting cytoplasmic channels. By staining vacuoles with a fluorescent dye and constructing 3D images of chloroplasts, and by microinjecting a fluorescence dye into the vacuole of living cells, it was demonstrated that the mature cell has only one vacuole, which is traversed by cytoplasmic channels connecting the CCC with the PCC. Immunofluorescent studies on isolated chlorenchyma cells treated with cytoskeleton disrupting drugs suspended in different levels of osmoticum showed that both microtubules and actin filaments are important in maintaining the cytoplasmic domains. With prolonged exposure of plants to dim light, the cytoskeleton undergoes changes and there is a dramatic shift of the CCC from the center toward the distal end of the cell.     

Taxonomic and ecological aspects of the distribution of glycinebetaine and related compounds in plants

Summary The concentrations of the major inorganic ions and glycinebetaine, choline and proline and the osmotic pressure of extract sap have been determined in eight salt marsh species and four sand dune species from local habitats. These results together with those previously reported on hydroponically grown plants and data assembled from the literature show that glycinebetaine accumulation is a feature of members of the Chenopodiaceae, Amaranthaceae, many Gramineae and some members of the Solanaceae and Compositae, particularly when exposed to conditions of low soil water potential. It is suggested that in these families betaine is employed as a non-toxic cytoplasmic osmoticum when decreased osmotic potentials are required. In some other plant species proline may fulfil a similar function. Another quaternary ammonium compound may be accumulated in the Plumbaginaceae in addition to proline. Some evidence suggests that the differences in the organic osmoticum used may relate to the different inorganic ion contents of the plants. The accumulation of nitrogen dipoles as cytoplasmic osmotica may make heavy demands on the nitrogen economy of the plants and this problem is discussed briefly.     

Genetic engineering of the biosynthesis of glycinebetaine enhances thermotolerance of photosystem II in tobacco plants

Genetically engineered tobacco (Nicotiana tabacum L.) with the ability to accumulate glycinebetaine was established. The wild type and transgenic plants were exposed to heat treatment (25–50°C) for 4 h in the dark and under growth light intensity (300 μmol m⁻² s⁻¹). The analyses of oxygen-evolving activity and chlorophyll fluorescence demonstrated that photosystem II (PSII) in transgenic plants showed higher thermotolerance than in wild type plants in particular when heat stress was performed in the light, suggesting that the accumulation of glycinebetaine leads to increased tolerance to heat-enhanced photoinhibition. This increased tolerance was associated with an improvement on thermostability of the oxygen-evolving complex and the reaction center of PSII. The enhanced tolerance was caused by acceleration of the repair of PSII from heat-enhanced photoinhibition. Under heat stress, there was a significant accumulation of H₂O₂, O₂⁻ and catalytic Fe in wild type plants but this accumulation was much less in transgenic plants. Heat stress significantly decreased the activities of catalase, ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and monodehydroascorbate reductase in wild type plants whereas the activities of these enzymes either decreased much less or maintained or even increased in transgenic plants. In addition, heat stress increased the activity of superoxide dismutase in wild type plants but this increase was much greater in transgenic plants. Furthermore, transgenic plants also showed higher content of ascorbate and reduced glutathione than that of wild type plants under heat stress. The results suggest that the increased thermotolerance induced by accumulation of glycinebetaine in vivo was associated with the enhancement of the repair of PSII from heat-enhanced photo inhibition, which might be due to less accumulation of reactive oxygen species in transgenic plants.     

Accumulation of glycinebetaine and its synthesis from radioactive precursors under salt-stress in the cyanobacterium Aphanothece halophytica

Growth in salt-stressed (2.0 M NaCl) Aphanothece halophytica was initially delayed during the first two days of cultivation and eventually attained the same growth rate as the control (0.5 M NaCl) cells. Glycinebetaine accumulation increased slightly in control cells but a dramatic increase of glycinebetaine occurred in salt-stressed cells during a growth period of six days. There was no apparent increase in the synthesis of [¹⁴C] glycinebetaine in the control cells, in contrast to the marked increase in its synthesis in the salt-stressed cells. Increasing NaCl concentration in the growth medium induced both the accumulation and the synthesis of glycinebetaine. Time course experiments provided evidence that [¹⁴C] choline was first oxidized to [¹⁴C] betaine aldehyde which was further oxidized to [¹⁴C] glycinebetaine in A. halophytica. The supporting data for such a pathway were obtained from the presence of choline and betaine aldehyde dehydrogenase activities found in the membrane and cytoplasmic fractions, respectively. The activities of these two enzymes were also enhanced upon increasing NaCl concentration in the growth medium from 0.5 M to 2.0 M. Under this condition an increaseof approximately 1.5-fold was observed for choline dehydrogenase activity as compared to 2.5-fold for betaine aldehyde dehydrogenase activity, suggesting a preferable induction of the latter enzyme by salt stress. A. halophytica was able to utilize [¹⁴C] ethanolamine and [¹⁴C] glycine for the synthesis of [¹⁴C] glycinebetaine. This revised version was published online in August 2006 with corrections to the Cover Date.     

逆境条件下植物体内渗透调节物质的积累与活性氧代谢

本文介绍了逆境迫下植物体内渗透调节物质的积累和作用,及其对活性氧的产生与清除的影响,阐述以脯氨酸为代表的渗透调节物质对活性氧的直接清除作用,Ca^2 ,甜菜碱等对抗氧化酶活性及抗氧化剂含量的影响,近年来人们广泛利用转基因技术合成氨酸,甜菜碱,为提高作物的抗氧化能车及培育抗逆新品种提供了一条有效途径。     

Response to chilling of tomato mesophyll protoplasts

Freshly isolated protoplasts from tomato leaves show two completely different responses to a chilling treatment of 12 h at 7° C prior to culture at 29° C, depending on the presence or absence of glucose in the medium. In the culture medium with glucose as osmoticum, where the rate of cell divisions under optimal culture conditions is relatively high (about 20% plating efficiency), protoplasts were drastically injured by the chilling procedure and died. In the medium with mannitol as the osmoticum instead of glucose, where the plating efficiency even under optimal conditions is rather low (about 8%), protoplasts withstand the chilling procedure. More-over, after the chilling treatment when the protoplasts were transferred to the optimal culture temperature of 29° C, the plating efficiency was raised to about 20%, which is the same level as in the glucose-containing medium without chilling. This effect was not observed when the medium in which the protoplasts were suspended during the chilling period was replaced with fresh medium. This suggests that under these conditions tomato protoplasts produce and excrete a factor in the cold that improves the vitality of the cells or stimulates cell division. The possible relationship between chilling sensitivity of tomato protoplasts and their ability to divide will be discussed.     

Transformation of Brassica juncea (L.) Czern with bacterial codA gene enhances its tolerance to salt stress

The codA gene for biosynthesis of glycinebetaine from Arthrobacter globiformis was used for transforming Brassica juncea cv. Pusa Jaikisan (which lack any means to synthesize glycinebetaine) through Agrobacterium mediated transformation. The stable insertion of the codA gene in the shoots obtained on medium with kanamycin and hygromycin was confirmed by PCR analysis of the nptII gene. Southern hybridization with a codA probe further demonstrated its successful integration. Immunoblot analysis revealed the presence of choline oxidase demonstrating that the bacterial codA gene had been successfully transcribed and translated. The seeds of transgenic lines showed enhanced capacity to germinate under salt stress as compared to that of the wild type. Further, the seedlings of transgenic plants that expressed codA gene showed significantly higher growth than that of the wild type under salt stress conditions. These results demonstrated that the introduction of a biosynthetic pathway for glycinebetaine into Brassica juncea significantly enhanced their salt tolerance. Hence, homozygous genotypes of selected transformed lines can be exploited for improving the salt tolerance of the desirable cultivars of Brassica juncea through breeding programmes.     

Manipulating the Length of the <Emphasis Type="Italic">b</Emphasis> Subunit F<Subscript>1</Subscript> Binding Domain in F<Subscript>1</Subscript>F<Subscript>0</Subscript> ATP Synthase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The peripheral stalk of F₁F₀ ATP synthase is composed of a parallel homodimer of b subunits that extends across the cytoplasmic membrane in F₀ to the top of the F₁ sector. The stalk serves as the stator necessary for holding F₁ against movement of the rotor. A series of insertions and deletions have been engineered into the hydrophilic domain that interacts with F₁. Only the hydrophobic segment from {val-121} to {ala-132} and the extreme carboxyl terminus proved to be highly sensitive to mutation. Deletions in either site apparently abolished enzyme function as a result of defects is assembly of the F₁F₀ complex. Other mutations manipulating the length of the sequence between these two areas had only limited effects on enzyme function. Expression of a b subunit with insertions with as few as two amino acids into the hydrophobic segment also resulted in loss of F₁F₀ ATP synthase. However, a fully defective b subunit with seven additional amino acids could be stabilized in a heterodimeric peripheral stalk within a functional F₁F₀ complex by a normal b subunit.     

Optimization of protoplast formation,regeneration, and viability in Microsporum gypseum

Factors affecting high yields, regeneration frequencies, and viability of protoplasts from clonal cultures of Microsporum gypseum were investigated. Maximum yields of protoplasts were obtained after 6 hrs digestion of 2–4 days old mycelium with Novozyme 234 using CaCl₂ (0.4 M) as an osmotic stabilizer and glycine + HCl (pH 4.5) as the buffer system. Mercaptoethanol + dithiothreitol (0.01 M) proved to be the best pretreatment of mycelium prior to digestion with enzyme. A regeneration frequency of 94.4% was obtained using the top agar method with complete medium (pH 6.5) containing 0.5% agar and 0.4 M CaCl₂ as an osmoticum. Colonies from regenerated protoplasts on medium containing CaCl₂ were pigmented and completely powdery with high sporulation. Protoplast viability was studied in osmotic stabilizer supplemented with glucose or glutamine. After 24 hrs, glucose (2%) and glutamine (2%) enhanced protoplast viability by 22% and 23%, respectively. Protein synthesis, as measured by ³H-lysine uptake, matched the viability profile determined by fluorescence microscopy.