Exploring the photosynthetic production capacity of sucrose by cyanobacteria

Because cyanobacteria are photosynthetic, fast-growing microorganisms that can accumulate sucrose under salt stress, they have a potential application as a sugar source for the biomass-derived production of renewable fuels and chemicals. In the present study, the production of sucrose by the cyanobacteria Synechocystis sp. PCC6803, Synechococcus elongatus PCC7942, and Anabaena sp. PCC7120 was examined. The three species displayed different growth curves and intracellular sucrose accumulation rates in response to NaCl. Synechocystis sp. PCC6803 was used to examine the impact of modifying the metabolic pathway on the levels of sucrose production. The co-overexpression of sps (slr0045), spp (slr0953), and ugp (slr0207) lead to a 2-fold increase in intracellular sucrose accumulation, whereas knockout of ggpS (sll1566) resulted in a 1.5-fold increase in the production of this sugar. When combined, these genetic modifications resulted in a fourfold increase in intracellular sucrose accumulation. To explore methods for optimizing the transport of the intracellular sucrose to the growth medium, the acid-wash technique and the CscB (sucrose permease)-dependent export method were evaluated using Synechocystis sp. PCC6803. Whereas the acid-wash technique proved to be effective, the CscB-dependent export method was not effective. Taken together, these results suggest that using genetic engineering, photosynthetic cyanobacteria can be optimized for efficient sucrose production.     

The silver lining of a viral agent: increasing seed yield and harvest index in Arabidopsis by ectopic expression of the potato leaf roll virus movement protein

Ectopic expression of viral movement proteins (MPs) has previously been shown to alter plasmodesmata (PD) function and carbon partitioning in transgenic plants, giving rise to the view of PD being dynamic and highly regulated structures that allow resource allocation to be adapted to environmental and developmental needs. However, most work has been restricted to solanaceous species and the potential use of MP expression to improve biomass and yield parameters has not been addressed in detail. Here we demonstrate that MP-mediated modification of PD function can substantially alter assimilate allocation, biomass production, and reproductive growth in Arabidopsis (Arabidopsis thaliana). These effects were achieved by constitutive expression of the potato leaf roll virus 17-kD MP (MP17) fused to green fluorescent protein (GFP) in different Arabidopsis ecotypes. The resulting transgenic plants were analyzed for PD localization of the MP17:GFP fusion protein and different lines with low to high expression levels were selected for further analysis. Low-level accumulation of MP17 resulted in enhanced sucrose efflux from source leaves and a considerably increased vegetative biomass production. In contrast, high MP17 levels impaired sucrose export, resulting in source leaf-specific carbohydrate accumulation and a strongly reduced vegetative growth. Surprisingly, later during development the MP17-mediated inhibition of resource allocation was reversed, and final seed yield increased in average up to 30% in different transgenic lines as compared to wild-type plants. This resulted in a strongly improved harvest index. The release of the assimilate export block was paralleled by a reduced PD binding of MP17 in senescing leaves, indicating major structural changes of PD during leaf senescence.     

Dynamics of root growth stimulation in Nicotiana tabacum in increasing light intensity

Light intensity is crucial for plant growth. In this study, the hypothesis was tested whether a sudden increase in light intensity leads to an immediate increase of root growth. Seedlings of Nicotiana tabacum grown in agar-filled Petri dishes were subjected to light intensities of 60 and 300 micromol m(-2) s(-1), respectively. Seedling biomass, sucrose, glucose and fructose concentration as well as primary root growth increased significantly with light intensity. The dynamics of the increase in root growth were analysed here in more detail. In transition experiments from low to high light intensities, root growth increased by a factor of four within 4 d, reaching the steady-state level measured in plants that were cultivated in high-light conditions. The distribution of relative elemental growth rates along the root growth zone retained a constant shape throughout this transition. During the first three hours after light increase, strong growth fluctuations were repeatedly observed with the velocity of the root tip cycling in a sinusoidal pattern between 120 and 180 microm h(-1). These dynamic patterns are discussed in the context of hydraulic and photosynthetic acclimation to the altered conditions. Experiments with externally applied sucrose and with transgenic plants having reduced capacities for sucrose synthesis indicated clearly that increasing light intensity rapidly enhanced root growth by elevating sucrose export from shoot to root.     

Magnesium deficiency in sugar beets alters sugar partitioning and phloem loading in young mature leaves

Magnesium deficiency has been reported to affect plant growth and biomass partitioning between root and shoot. The present work aims to identify how Mg deficiency alters carbon partitioning in sugar beet (Beta vulgaris L.) plants. Fresh biomass, Mg and sugar contents were followed in diverse organs over 20 days under Mg-sufficient and Mg-deficient conditions. At the end of the treatment, the aerial biomass, but not the root biomass, of Mg-deficient plants was lower compared to control plants. A clear inverse relationship between Mg and sugar contents in leaves was found. Mg deficiency promoted a marked increase in sucrose and starch accumulation in the uppermost expanded leaves, which also had the lowest content of Mg among all the leaves of the rosette. The oldest leaves maintained a higher Mg content. [¹⁴C]Sucrose labelling showed that sucrose export from the uppermost expanded leaves was inhibited. In contrast, sucrose export from the oldest leaves, which are close to, and export mainly to, the roots, was not restricted. In response to Mg deficiency, the BvSUT1 gene encoding a companion cell sucrose/H⁺ symporter was induced in the uppermost expanded leaves, but without further enhancement of sucrose loading into the phloem. The observed increase in BvSUT1 gene expression supports the idea that sucrose loading into the phloem is defective, resulting in its accumulation in the leaf.     

Biochemical basis for effects of k-deficiency on assimilate export rate and accumulation of soluble sugars in soybean leaves

The effects of K-deficiency on carbon exchange rates (CER), photosynthate partitioning, export rate, and activities of key enzymes involved in sucrose metabolism were studied in soybean (Glycine max [L.] Merr.) leaves. The different parameters were monitored in mature leaves that had expanded prior to, or during, imposition of a complete K-deficiency (plants received K-free nutrition solution). In general, recently expanded leaves had the highest concentration of K, and imposition of K-stress at any stage of leaf expansion resulted in decreased K concentrations relative to control plants (10 millimolar K). A reduction in CER, relative to control plants, was only observed in leaves that expanded during the K-stress. Stomatal conductance also declined, but this was not the primary cause of the decrease in carbon fixation because internal CO₂ concentration was unaffected by K-stress. Assimilate export rate from K-deficient leaves was reduced but relative export, calculated as a percentage of CER, was similar to control leaves. Over all the data, export rate was correlated positively with both CER and activity of sucrose phosphate synthase in leaf extracts. K-deficient leaves had higher concentrations of sucrose and hexose sugars. Accumulation of hexose sugars was associated with increased activities of acid invertase. Neutral invertase activity was low and unaffected by K-nutrition. It is concluded that decreased rates of assimilate export are associated with decreased activities of sucrose phosphate synthase, a key enzyme involved in sucrose formation, and that accumulation of hexose sugars may occur because of increased hydrolysis of sucrose in K-deficient leaves.     

Study of the production of fructose and ethanol from sucrose media by Saccharomyces cerevisiae

The production of ethanol and enriched fructose syrups from a synthetic medium with various sucrose concentrations using the mutant Saccharomyces cerevisiae ATCC 36858 was investigated. In batch tests, fructose yields were above 90% of theoretical values for the sucrose concentrations between 35 g/l and 257 g/l. The specific growth rates and biomass yields were from 0.218 to 0.128 h(-1) and from 0.160 to 0.075 g biomass/g of glucose and fructose consumed, respectively. Ethanol yields were in the range of 72 to 85% of theoretical value when sucrose concentrations were above 81 g/l. The volumetric ethanol productivity was 2.23 g ethanol/(l h) in a medium containing 216 g/l sucrose. Fructo-oligosaccharides and glycerol were also produced in the process. A maximum fructo-oligosaccharides concentration (up to 9 g/l) was attained in the 257 g/l sucrose medium in the first 7 h of the fermentation. These sugars started to be consumed when the concentrations of sucrose in the media were less than 30% of its initial values. The fructo-oligosaccharides mixture was composed of 6-kestose (61.5%), neokestose (29.7%) and 1-kestose (8.8%). The concentration of glycerol produced in the process was less than 9 g/l. These results will be useful in the production of enriched fructose syrups and ethanol using sucrose-based raw materials.     

Double mutation in photosystem II reaction centers and elevated CO2 grant thermotolerance to mesophilic cyanobacterium

Photosynthetic biomass production rapidly declines in mesophilic cyanobacteria grown above their physiological temperatures largely due to the imbalance between degradation and repair of the D1 protein subunit of the heat susceptible Photosystem II reaction centers (PSIIRC). Here we show that simultaneous replacement of two conserved residues in the D1 protein of the mesophilic Synechocystis sp. PCC 6803, by the analogue residues present in the thermophilic Thermosynechococcus elongatus, enables photosynthetic growth, extensive biomass production and markedly enhanced stability and repair rate of PSIIRC for seven days even at 43 °C but only at elevated CO(2) (1%). Under the same conditions, the Synechocystis control strain initially presented very slow growth followed by a decline after 3 days. Change in the thylakoid membrane lipids, namely the saturation of the fatty acids is observed upon incubation for the different strains, but only the double mutant shows a concomitant major change of the enthalpy and entropy for the light activated Q(A)(-)→Q(B) electron transfer, rendering them similar to those of the thermophilic strain. Following these findings, computational chemistry and protein dynamics simulations we propose that the D1 double mutation increases the folding stability of the PSIIRC at elevated temperatures. This, together with the decreased impairment of D1 protein repair under increased CO(2) concentrations result in the observed photothermal tolerance of the photosynthetic machinery in the double mutant.     

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.     

Influence of auxins and sucrose in monoterpenoid oxindole alkaloid production by Uncaria tomentosa cell suspension cultures

Growth and alkaloid production in Uncaria tomentosa cell suspension cultures were studied in Murashige and Skoog medium supplemented with 10 microM 2,4-dichlorophenoxyacetic acid, 10 microM kinetin, and 58 mM sucrose for maintenance and with 10 microM indole-3-acetic acid, 10 microM kinetin, and 58 mM sucrose for production. A U. tomentosa pale Uth-3 cell line, cultured in the production medium, showed a reduced lag phase and a specific growth rate (mu) of 0.27 day(-1), while cells growing in the maintenance medium showed mu = 0.20 day(-1). U. tomentosa cells growing in the production medium produced monoterpenoid oxindole alkaloids (MOA) in amounts of 10.2 +/- 1.6 microg g(-1) dry weight (DW). The chemical profile of MOA produced by in vitro cell cultures was similar to that found in the plant. After 10 subcultures, maximum MOA production decreased to 2.0 +/- 0.7 microg g(-1) DW, while tryptamine alkaloids (TA) were produced with a maximum of 6.2 +/- 0.4 microg g(-1) DW. The increase of initial sucrose concentration up to 145 mM in the production medium enhanced the cell biomass by 3.2-fold (from 10.2 +/- 0.1 to 32.8 +/- 1.1 g DW L(-1)), reduced mu from 0.27 to 0.23 day(-1), and provoked a substantial accumulation of TA (23.1 +/- 4.7 microg g(-1) DW). A high sucrose concentration stimulated MOA production in the maintenance medium (2.7 +/- 0.5 microg g(-1) DW), even in the presence of 2,4-dichlorophenoxyacetic acid.     

Functional differences of photosystem II from Synechococcus elongatus and spinach characterized by flash induced oxygen evolution patterns

Detailed comparative studies of flash induced oxygen evolution patterns in thylakoids from the thermophilic cyanobacterium Synechococcus elongatus (S. elongatus; also referred to as Thermosynechococcus elongatus) and from spinach led to the following results: (i) the miss parameter alpha of S. elongatus thylakoids exhibits a pronounced temperature dependence with a minimum of 7% at 25 degrees C and values of 17 and 10% at 3 and 35 degrees C, respectively, while for spinach thylakoids alpha decreases continuously from 18% at 35 degrees C down to 8% at 3 degrees C; (ii) at all temperatures, the double hit probability beta exceeds in S. elongatus the corresponding values of spinach by an increment Delta beta of about 3%; (iii) at 20 degrees C the slow relaxation of the oxidation states S(2) and S(3) is about 15 and 30 times, respectively, slower in S. elongatus than in spinach, while the reduction of these S states by tyrosine Y(D) is 2-3 times faster; (iv) the reaction S(0)Y(D)(ox) --> S(1)Y(D) is slower by a factor of 4 in S. elongatus as compared to spinach; and (v) the activation energies of S state dark relaxations in S. elongatus are all within a factor of 1.5 as compared to the previously reported values from spinach thylakoids [Vass, I., Deak, Z., and Hideg, E. (1990) Biochim. Biophys. Acta 1017, 63-69; Messinger, J., Schr?der, W. P., and Renger, G. (1993) Biochemistry 32, 7658-7668], but the difference between the activation energies of the slow S(2) and S(3) decays is significantly larger in S. elongatus than in spinach. These results are discussed in terms of differences between cyanobacteria and higher plants on the acceptor side of PSII and a shift of the redox potential of the couple Y(D)/Y(D)(ox). The obtained data are also suitable to address questions about effects of the redox state of Y(D) on the miss probability and the possibility of an S state dependent miss parameter.     

Nitric oxide-induced formation of the S-2 state in the oxygen-evolving complex of photosystem II from Synechococcus elongatus

In spinach photosystem II (PSII) membranes, the tetranuclear manganese cluster of the oxygen-evolving complex (OEC) can be reduced by incubation with nitric oxide at -30 degrees C to a state which is characterized by an Mn(2)(II, III) EPR multiline signal [Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V. (1998) Biochemistry 37, 3581-3587]. This state was recently assigned to the S(-)(2) state of the OEC [Schansker, G., Goussias, C., Petrouleas, V., and Rutherford, A. W. (2002) Biochemistry 41, 3057-3064]. On the basis of EPR spectroscopy and flash-induced oxygen evolution patterns, we show that a similar reduction process takes place in PSII samples of the thermophilic cyanobacterium Synechococcus elongatus at both -30 and 0 degrees C. An EPR multiline signal, very similar but not identical to that of the S(-)(2) state in spinach, was obtained with monomeric and dimeric PSII core complexes from S. elongatus only after incubation at -30 degrees C. The assignment of this EPR multiline signal to the S(-)(2) state is corroborated by measurements of flash-induced oxygen evolution patterns and detailed fits using extended Kok models. The small reproducible shifts of several low-field peak positions of the S(-)(2) EPR multiline signal in S. elongatus compared to spinach suggest that slight differences in the coordination geometry and/or the ligands of the manganese cluster exist between thermophilic cyanobacteria and higher plants.     

Immobilized cyanobacteria as a biofertilizer for rice crops; Intl. Conference on Applied Algology, Knysna, South Africa, April 1996.

N₂-fixing cyanobacteria (Anabaena azollae, symbiont strains) were immobilized in polyurethane foam and ammonia production by the cyanobacteria was investigated in the laboratory and rice field. The cyanobacterial symbiont, A. azollae - MPK-SK-AM-24 showed the highest growth rate and biomass production amongst the 5 isolates examined while A. azollae-AS-DS showed the highest nitrogenase activity followed by A. variabilis - SA₀ (wild type, non-symbiotic). Treatment of the foam-immobilized cyanobacteria with the systemic fungicide Bavistin stimulated nitrogenase activity while inhibiting glutamine synthetase (GS) activity. Free-living A. azollae-MPK-SK-AF-38, A. azollae - MPK-SK-AM-24 and A. azollae-MPK-SK-AM-27 excreted the highest amounts of ammonia into the growth medium; under foam - immobilized conditions the ammonia production increased further. Treatment of the foam - immobilized cyanobacteria with the fungicides Bavistin and Vitavax resulted in ammonia production at significantly higher rates. Rice seedlings (var. ADT 36) grown in the laboratory in conjunction with foam - immobilized A. azollae showed increased growth. A field experiment with paddy rice and foam - immobilized A. azollae strains indicated that the cyanobacteria excreted significant amounts of ammonia into the flood water in the rice fields resulting in increased chlorophyll content of the plants and increased the rice grain and straw yields. A combination of fertilizer nitrogen and inoculation with foam - immobilized cyanobacteria also significantly increased the rice grain and straw yield. Additionally, both A. azollae and A. variabilis were immobilized in sugarcane waste (bagasse), added to rice paddy and resulted in increased rice grain yield. This revised version was published online in September 2006 with corrections to the Cover Date.     

Characteristics of Sucrose Transport and Sucrose-Induced H+ Transport on the Tonoplast of Red Beet (Beta vulgaris L.) Storage Tissue

Sucrose-induced changes of the energization state of the red beet root (Beta vulgaris L. ssp. conditiva) vacuolar membrane were observed with the fluorescent dyes 6-chloro-9-{[4-(diethylamino)- 1-methylbutyl]-amino}-2-methoxyacridine dihydrochloride, as a pH monitor, and 9-amino-6-chloro-2-methoxyacridine (ACMA). Consequently, transient acidification of the surrounding suspension medium could be measured with a pH electrode. This signal was specific for Suc and was not seen for sorbitol, mannitol, or maltose. Sucrose-induced medium acidification was sensitive to the same inhibitors that were efficient in inhibiting sucrose transport, including the monoclonal antibodies TNP56-12 and C50-5-3. It was seen with vacuoles and vesicles energized with MgATP before sucrose was added but also with vacuoles not artificially energized previously. Using bafilomycin A1 for the inhibition of the vacuolar ATPase of vacuoles previously energized by MgATP, apparent Km values for H+ export from the vacuoles to the medium could be calculated taking into account the passive proton leak. Apparent Km values for H+ export determined from data obtained with pH measurements in the medium and with ACMA corresponded to those obtained previously for sucrose uptake. Comparing sucrose uptake rates with corresponding H+ export rates at the respective sucrose concentrations and at Km, a stoichiometry of approximately one proton per transported sucrose was estimated.     

Effects of light and biomass partitioning on growth, photosynthesis and carbohydrate content of the seagrass Zostera noltii Hornem

Plants of the seagrass Zostera noltii were cultured in the laboratory (mesocosms) for two weeks to assess the effect of above:below-ground (AG/BG) biomass ratios and light on growth, photosynthesis and chemical composition. Experimental plant units (EPUs) with different proportions between AG and BG biomass were obtained from plants of the same size (containing 6 shoots and 5 internodes) by excising 0-5 shoots. The EPUs maintained the proportions in AG/BG biomass ratios during the experiment. While growth rate was unaffected by biomass partitioning at high light, maximum growth at low light was recorded in plants with low AG/BG ratios. The production of shoots and rhizomes showed a compensatory morphological response depending on the initial AG/BG proportions regardless of the light level. While shoot production, estimated as shoot appearance rate, was high at low AG/BG ratios and minimal under high AG/BG values, rhizome production, estimated as internode appearance rate and internode elongation rate, was maximal under high AG/BG proportions and decreased towards lower AG/BG ratios. This rhizomatic response was observed for secondary rhizomes and not for primary ones. In contrast to morphological response, no significant differences were detected in maximum electron transport rates (ETRm) among the different shoots in the plant. However, mean values of ETRm in plants were affected by biomass partitioning and light. EPUs grown in low light increased the sucrose stored in shoots as the AG/BG biomass ratios decreased; however, EPUs grown at high light showed no effect of biomass partitioning on sucrose levels. In conclusion, shoots excision by experimental manipulation caused a compensatory morphological response in plants while photosynthetic performance remained almost unaffected.     

L-amino acid oxidases with specificity for basic L-amino acids in cyanobacteria

The two closely related fresh water cyanobacteria Synechococcus elongatus PCC 6301 and Synechococcus elongatus PCC 7942 have previously been shown to constitutively express a FAD-containing L-amino acid oxidase with high specificity for basic L-amino acids (L-arginine being the best substrate). In this paper we show that such an enzyme is also present in the fresh water cyanobacterium Synechococcus cedrorum PCC 6908. In addition, an improved evaluation of the nucleotide/amino acid sequence of the L-amino acid oxidase of Synechococcus elongatus PCC 6301 (encoded by the aoxA gene) with respect to the FAD-binding site and a translocation pathway signal sequence will be given. Moreover, the genome sequences of 24 cyanobacteria will be evaluated for the occurrence of an aoxA-similar gene. In the evaluated cyanobacteria 15 genes encoding an L-amino acid oxidase-similar protein will be found.     

Extinction coefficients of cytochromes b559 and c550 of Thermosynechococcus elongatus and Cyt b559/PS II stoichiometry of higher plants

"Reduced minus oxidized" difference extinction coefficients Deltavarepsilon in the alpha-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1+/-1.0 mM(-1) cm(-1) and 27.0+/-1.0 mM(-1) cm(-1) were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from -250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E(m) values at pH 6.5 of 244+/-11 mV and -94+/-21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the alpha-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q(B) site and the microenvironment of the heme group of Cyt b559 are discussed.     

Environmental forcing of nitrogen fixation in the eastern tropical and sub-tropical North Atlantic Ocean

During the winter of 2006 we measured nifH gene abundances, dinitrogen (N(2)) fixation rates and carbon fixation rates in the eastern tropical and sub-tropical North Atlantic Ocean. The dominant diazotrophic phylotypes were filamentous cyanobacteria, which may include Trichodesmium and Katagnymene, with up to 10(6) L(-1)nifH gene copies, unicellular group A cyanobacteria with up to 10(5) L(-1)nifH gene copies and gamma A proteobacteria with up to 10(4) L(-1)nifH gene copies. N(2) fixation rates were low and ranged between 0.032-1.28 nmol N L(-1) d(-1) with a mean of 0.30 ± 0.29 nmol N L(-1) d(-1) (1σ, n = 65). CO(2)-fixation rates, representing primary production, appeared to be nitrogen limited as suggested by low dissolved inorganic nitrogen to phosphate ratios (DIN:DIP) of about 2 ± 3.2 in surface waters. Nevertheless, N(2) fixation rates contributed only 0.55 ± 0.87% (range 0.03-5.24%) of the N required for primary production. Boosted regression trees analysis (BRT) showed that the distribution of the gamma A proteobacteria and filamentous cyanobacteria nifH genes was mainly predicted by the distribution of Prochlorococcus, Synechococcus, picoeukaryotes and heterotrophic bacteria. In addition, BRT indicated that multiple a-biotic environmental variables including nutrients DIN, dissolved organic nitrogen (DON) and DIP, trace metals like dissolved aluminum (DAl), as a proxy of dust inputs, dissolved iron (DFe) and Fe-binding ligands as well as oxygen and temperature influenced N(2) fixation rates and the distribution of the dominant diazotrophic phylotypes. Our results suggest that lower predicted oxygen concentrations and higher temperatures due to climate warming may increase N(2) fixation rates. However, the balance between a decreased supply of DIP and DFe from deep waters as a result of more pronounced stratification and an enhanced supply of these nutrients with a predicted increase in deposition of Saharan dust may ultimately determine the consequences of climate warming for N(2) fixation in the North Atlantic.     

High efficiency plant production of North American ginseng via somatic embryogenesis from cotyledon explants

An efficient in vitro protocol for plant production of North American ginseng has been established. The pretreatment of cotyledon explants with 1.0 M sucrose at 4 degrees C resulted in an improvement of embryo quality and, combined with a higher sucrose content (7%) in induction medium, improved the embryogenesis frequency from 40% to 75% and the number of embryos per explant from 10 to 21. The frequency of secondary embryogenesis from somatic embryo-derived tissues cultured on MS medium with 1.0 mg l(-1) 2, 4-D and 1.0 mg l(-1) NAA is up to 90%. Somatic embryos can further develop to maturity on SH medium supplemented with 1% activated charcoal and half of them can germinate. About 85% of the germinated embryos will convert into plants with well-developed taproot systems on 1/2 SH medium with 0.5% activated charcoal. The growth chamber and field establishment rates were 95.6 and 93.7%, respectively. The plants transplanted to growth chambers and field plots appear normal.     

Sucrose export defective1 encodes a novel protein implicated in chloroplast-to-nucleus signaling

The Sucrose export defective1 (Sxd1) gene of maize was cloned and shown to encode a novel protein conserved between plants and cyanobacteria. The structure of the Sxd1 locus was determined in wild-type plants and two independent sxd1 alleles. Expression analysis demonstrated that the gene was transcribed in all green tissues, with highest levels in maturing leaf blades. In situ hybridization studies revealed high levels of Sxd1 mRNA in bundle sheath cells, with lower levels within the mesophyll. The SXD1 protein was localized to chloroplasts, in both bundle sheath and mesophyll cells. Levels of sucrose, glucose, and fructose were compared between wild-type and sxd1 plants. Mutant plants were fully capable of producing sucrose and accumulated all three sugars at concentrations above those measured in wild-type plants. Despite these increased sugar concentrations, photosynthetic gene expression was not significantly downregulated in affected areas of sxd1 leaf blades. These results are consistent with photosynthate being trapped within anthocyanin-accumulating regions of sxd1 leaves due to plasmodesmal occlusion at the bundle sheath-vascular parenchyma boundary of the minor veins. A model for SXD1 function is proposed in which the protein is involved in a chloroplast-to-nucleus signaling pathway necessary for proper late-stage differentiation of maize bundle sheath cells, including the developmentally regulated modification of plasmodesmata.