The role of ecotypic variation in driving worldwide colonization by a cosmopolitan plant.

For almost 100 years now, ecotypic differentiation of plant species has been a major topic of research. In changing environments, the question needs to be answered as to how long it takes to adapt, and which parameters are subject to this fast adaptation. Short-living colonizing plant species are excellent examples, especially when they are selfing. Shepherd's Purse Capsella bursa-pastoris (Brassicaceae) is one of the most wide-spread flowering species on earth and avoids only the hot and humid tropics. Many studies demonstrated the ecotypic differentiation of C. bursa-pastoris in various regions of the world but ecotypic differentiation regarding adaptability of anatomy and physiology of rosette leaves so far remained less recognized. However, the leaves are relevant for subsequent seed set; in particular, winter-annual accessions require a robust rosette to survive adverse conditions. Leaf-related traits such as the thickness of the mesophyll and epidermis, stomatal density, photosynthetic capacity and the ability to withstand and even use high light conditions were therefore analysed in provenances from various climatic zones. Photosynthetic capacity depends on leaf anatomy and cellular physiological parameters. In particular, the ability to dynamically adjust the photosynthetic capacity to changing environmental conditions results in higher fitness. Here, we attempt to relate these results to the four Mendelian leaf types according to Shull.

Rhizoremediation of diesel-contaminated soil with two rapeseed varieties and petroleum degraders reveals different responses of the plant defense mechanisms.

Plant-assisted bioremediation (rhizoremediation) stands out as a potential tool to inactivate or completely remove xenobiotics from the polluted environment. Therefore, it is of key importance to find an adequate combination of plant species and microorganisms that together enhance the clean-up process. To understand the response of plants upon bioaugmentation, the antioxidative and detoxification system was analyzed in high and low erucic acid rapeseed varieties (HEAR and LEAR, respectively), after 8 weeks of their treatment with petroleum degraders and 6000 mg diesel oil/kg dry soil. The oxidative stress was enhanced in LEAR being exposed to sole diesel oil, in comparison with HEAR. However, when LEAR plants were additionally inoculated with bacteria, suppression of total catalase (CAT) and ascorbate peroxidase (APX) activity were observed. Interestingly, glutathione transferase (GST) activity was found in these plants at a much higher level than in HEAR, which correlated with a more efficient diesel removal performed by LEAR in the polluted soil and upon bioaugmentation. A distinct profile of polycyclic aromatic hydrocarbons (PAH) was detected in leaves of these plants. Neither LEAR nor HEAR experienced any changes in the photosynthetic capacity upon diesel pollution and presence of petroleum degraders, which supports the usefulness of rhizoremediation with rapeseed.

Expression of the minor isoform pea ferredoxin in tobacco alters photosynthetic electron partitioning and enhances cyclic electron flow.

Ferredoxins (Fds) are ferrosulfoproteins that function as low-potential electron carriers in plants. The Fd family is composed of several isoforms that share high sequence homology but differ in functional characteristics. In leaves, at least two isoforms conduct linear and cyclic photosynthetic electron transport around photosystem I, and mounting evidence suggests the existence of at least partial division of duties between these isoforms. To evaluate the contribution of different kinds of Fds to the control of electron fluxes along the photosynthetic electron transport chain, we overexpressed a minor pea (Pisum sativum) Fd isoform (PsFd1) in tobacco (Nicotiana tabacum) plants. The transplastomic OeFd1 plants exhibited variegated leaves and retarded growth and developmental rates. Photosynthetic studies of these plants indicated a reduction in carbon dioxide assimilation rates, photosystem II photochemistry, and linear electron flow. However, the plants showed an increase in nonphotochemical quenching, better control of excitation pressure at photosystem II, and no evidence of photoinhibition, implying a better dynamic regulation to remove excess energy from the photosynthetic electron transport chain. Finally, analysis of P700 redox status during illumination confirmed that the minor pea Fd isoform promotes enhanced cyclic flow around photosystem I. The two novel features of this work are: (1) that Fd levels achieved in transplastomic plants promote an alternative electron partitioning even under greenhouse light growth conditions, a situation that is exacerbated at higher light intensity measurements; and (2) that an alternative, minor Fd isoform has been overexpressed in plants, giving new evidence of labor division among Fd isoforms.

N-terminal structure of maize ferredoxin:NADP+ reductase determines recruitment into different thylakoid membrane complexes.

To adapt to different light intensities, photosynthetic organisms manipulate the flow of electrons through several alternative pathways at the thylakoid membrane. The enzyme ferredoxin:NADP(+) reductase (FNR) has the potential to regulate this electron partitioning because it is integral to most of these electron cascades and can associate with several different membrane complexes. However, the factors controlling relative localization of FNR to different membrane complexes have not yet been established. Maize (Zea mays) contains three chloroplast FNR proteins with totally different membrane association, and we found that these proteins have variable distribution between cells conducting predominantly cyclic electron transport (bundle sheath) and linear electron transport (mesophyll). Here, the crystal structures of all three enzymes were solved, revealing major structural differences at the N-terminal domain and dimer interface. Expression in Arabidopsis thaliana of maize FNRs as chimeras and truncated proteins showed the N-terminal determines recruitment of FNR to different membrane complexes. In addition, the different maize FNR proteins localized to different thylakoid membrane complexes on expression in Arabidopsis, and analysis of chlorophyll fluorescence and photosystem I absorbance demonstrates the impact of FNR location on photosynthetic electron flow.

Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase.

The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C(3) plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck-Halliwell-Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants.

Cyanobacterial flavodoxin complements ferredoxin deficiency in knocked-down transgenic tobacco plants.

Ferredoxins are the main electron shuttles in chloroplasts, accepting electrons from photosystem I and delivering them to essential oxido-reductive pathways in the stroma. Ferredoxin levels decrease under adverse environmental conditions in both plants and photosynthetic micro-organisms. In cyanobacteria and some algae, this decrease is compensated for by induction of flavodoxin, an isofunctional flavoprotein that can replace ferredoxin in many reactions. Flavodoxin is not present in plants, but tobacco lines expressing a plastid-targeted cyanobacterial flavodoxin developed increased tolerance to environmental stress. Chloroplast-located flavodoxin interacts productively with endogenous ferredoxin-dependent pathways, suggesting that its protective role results from replacement of stress-labile ferredoxin. We tested this hypothesis by using RNA antisense and interference techniques to decrease ferredoxin levels in transgenic tobacco. Ferredoxin-deficient lines showed growth arrest, leaf chlorosis and decreased CO(2) assimilation. Chlorophyll fluorescence measurements indicated impaired photochemistry, over-reduction of the photosynthetic electron transport chain and enhanced non-photochemical quenching. Expression of flavodoxin from the nuclear or plastid genome restored growth, pigment contents and photosynthetic capacity, and relieved the electron pressure on the electron transport chain. Tolerance to oxidative stress also recovered. In the absence of flavodoxin, ferredoxin could not be decreased below 45% of physiological content without fatally compromising plant survival, but in its presence, lines with only 12% remaining ferredoxin could grow autotrophically, with almost wild-type phenotypes. The results indicate that the stress tolerance conferred by flavodoxin expression in plants stems largely from functional complementation of endogenous ferredoxin by the cyanobacterial flavoprotein.

FdC1, a novel ferredoxin protein capable of alternative electron partitioning, increases in conditions of acceptor limitation at photosystem I.

In higher plants, [2Fe-2S] ferredoxin (Fd) proteins are the unique electron acceptors from photosystem I (PSI). Fds are soluble, and distribute electrons to many enzymes, including Fd:NADP(H) reductase (FNR), for the photoreduction of NADP(+). In addition to well studied [2Fe-2S] Fd proteins, higher plants also possess genes for significantly different, as yet uncharacterized Fd proteins, with extended C termini (FdCs). Whether these FdC proteins function as photosynthetic electron transfer proteins is not known. We examined whether these proteins play a role as alternative electron acceptors at PSI, using quantitative RT-PCR to follow how their expression changes in response to acceptor limitation at PSI, in mutant Arabidopsis plants lacking 90-95% of photosynthetic [2Fe-2S] Fd. Expression of the gene encoding one FdC protein, FdC1, was identified as being strongly up-regulated. We confirmed that this protein was chloroplast localized and increased in abundance on PSI acceptor limitation. We purified the recombinant FdC1 protein, which exhibited a UV-visible spectrum consistent with a [2Fe-2S] cluster, confirmed by EPR analysis. Measurements of electron transfer show that FdC1 is capable of accepting electrons from PSI, but cannot support photoreduction of NADP(+). Whereas FdC1 was capable of electron transfer with FNR, redox potentiometry showed that it had a more positive redox potential than photosynthetic Fds by around 220 mV. These results indicate that FdC1 electron donation to FNR is prevented because it is thermodynamically unfavorable. Based on our data, we speculate that FdC1 has a specific function in conditions of acceptor limitation at PSI, and channels electrons away from NADP(+) photoreduction.

Induction of the AOX1D isoform of alternative oxidase in A. thaliana T-DNA insertion lines lacking isoform AOX1A is insufficient to optimize photosynthesis when treated with antimycin A.

Plant respiration is characterized by two pathways for electron transfer to O(2), namely the cytochrome pathway (CP) that is linked to ATP production, and the alternative pathway (AP), where electrons from ubiquinol are directly transferred to O(2) via an alternative oxidase (AOX) without concomitant ATP production. This latter pathway is well suited to dispose of excess electrons in the light, leading to optimized photosynthetic performance. We have characterized T-DNA-insertion mutant lines of Arabidopsis thaliana that do not express the major isoform, AOX1A. In standard growth conditions, these plants did not show any phenotype, but restriction of electron flow through CP by antimycin A, which induces AOX1A expression in the wild-type, led to an increased expression of AOX1D in leaves of the aox1a-knockout mutant. Despite the increased presence of the AOX1D isoform in the mutant, antimycin A caused inhibition of photosynthesis, increased ROS, and ultimately resulted in amplified membrane leakage and necrosis when compared to the wild-type, which was only marginally affected by the inhibitor. It thus appears that AOX1D was unable to fully compensate for the loss of AOX1A when electron flow via the CP is restricted. A combination of inhibition studies, coupled to metabolite profiling and targeted expression analysis of the P-protein of glycine decarboxylase complex (GDC), suggests that the aox1a mutants attempt to increase their capacity for photorespiration. However, given their deficiency, it is intriguing that increase in expression neither of AOX1D nor of GDC could fully compensate for the lack of AOX1A to optimize photosynthesis when treated with antimycin A. We suggest that the aox1a mutants can further be used to substantiate the current models concerning the influence of mitochondrial redox on photosynthetic performance and gene expression.

Knockout of major leaf ferredoxin reveals new redox-regulatory adaptations in Arabidopsis thaliana.

Ferredoxins are the major distributors for electrons to the various acceptor systems in plastids. In green tissues, ferredoxins are reduced by photosynthetic electron flow in the light, while in heterotrophic tissues, nicotinamide adenine dinucleotide (reduced) (NADPH) generated in the oxidative pentose-phosphate pathway (OPP) is the reductant. We have used a Ds-T-DNA insertion line of Arabidopsis thaliana for the gene encoding the major leaf ferredoxin (Fd2, At1g60950) to create a situation of high electron pressure in the thylakoids. Although these plants (Fd2-KO) possess only the minor fraction of leaf Fd1 (At1g10960), they grow photoautotrophically on soil, but with a lower growth rate and less chlorophyll. The more oxidized conditions in the stroma due to the formation of reactive oxygen species are causing a re-adjustment of the redox state in these plants that helps them to survive even under high light. Redox homeostasis is achieved by regulation at both, the post-translational and the transcriptional level. Over-reduction of the electron transport chain leads to increased transcription of the malate-valve enzyme NADP-malate dehydrogenase (MDH), and the oxidized stroma leads to an increased transcription of the OPP enzyme glucose-6-P dehydrogenase. In isolated spinach chloroplasts, oxidized conditions give rise to a decreased activation state of NADP-MDH and an activation of glucose-6-P dehydrogenase even in the light. In Fd2-KO plants, NADPH-requiring antioxidant systems are upregulated. These adjustments must be caused by plastid signals, and they prevent oxidative damage under rather severe conditions.

Identification and localization of cyanophycin in bacteria cells via imaging of the nitrogen distribution using energy-filtering transmission electron microscopy.

In this study the technique of energy-filtering transmission electron microscopy was applied to localize cyanophycin (CGP) in recombinant strains of Ralstonia eutropha. Since CGP is a polymer consisting of the amino acids aspartate and arginine, which functions as a temporary nitrogen reserve that is deposited as insoluble inclusions in the cytoplasm of the cell, its nitrogen content is significantly higher than that of the other cell matter. In this study, we recorded nitrogen distribution maps, which represent the location of CGP in ultrathin sections of resin-embedded cells of recombinant strains of R. eutropha expressing the cyanophycin synthetase of Anabaena sp. strain PCC 7120. Furthermore, the existence of nitrogen in CGP granules was additionally proven by recording electron energy-loss spectra. The samples of R. eutropha H16 (pBBR1MCS-2::cphA1(7120)) revealed a second type of granule, which does not show nitrogen in the corresponding maps and which can be identified as an inclusion containing poly(3-hydroxybutyric acid). The methods applied in this study are suitable to identify storage compounds with elevated nitrogen contents and to reveal their location in the bacterial cell. The methods are also very helpful to distinguish between inclusions of different chemical compositions that occur both at the same time in the cells but cannot or only hardly be distinguished by other methods.

Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16.

The H(2)-oxidizing lithoautotrophic bacterium Ralstonia eutropha H16 is a metabolically versatile organism capable of subsisting, in the absence of organic growth substrates, on H(2) and CO(2) as its sole sources of energy and carbon. R. eutropha H16 first attracted biotechnological interest nearly 50 years ago with the realization that the organism's ability to produce and store large amounts of poly[R-(-)-3-hydroxybutyrate] and other polyesters could be harnessed to make biodegradable plastics. Here we report the complete genome sequence of the two chromosomes of R. eutropha H16. Together, chromosome 1 (4,052,032 base pairs (bp)) and chromosome 2 (2,912,490 bp) encode 6,116 putative genes. Analysis of the genome sequence offers the genetic basis for exploiting the biotechnological potential of this organism and provides insights into its remarkable metabolic versatility.

Optimization of cyanophycin production in recombinant strains of Pseudomonas putida and Ralstonia eutropha employing elementary mode analysis and statistical experimental design.

Elementary mode analysis was applied to simulate conditions for cyanophycin (CGP) biosynthesis and to optimize its production in bacteria. The conclusions from these simulations were confirmed by experiments with recombinant strains of the wild types and polyhydroxyalkanoate (PHA)-negative mutants of Ralstonia eutropha and Pseudomonas putida expressing CGP synthetase genes (cphA) of Synechocystis sp. strain PCC6308 or Anabaena sp. strain PCC7120. In particular, the effects of suitable precursor substrates and of oxygen supply as well as of the capability to accumulate PHA in addition to CGP biosynthesis were investigated. Since CGP consists of the amino acids aspartate and arginine, the tricarboxylic acid cycle (TCC), which provides intermediates for biosynthesis of these amino acids, seems to be important. Excretion of intermediates of the TCC upon cultivation at restricted oxygen supply and conversion of fumarate mainly to malate and to only little succinate in the absence of oxygen indicated that TCC intermediates for arginine and aspartate biosynthesis were provided by the oxidative or reductive parts of the TCC, respectively. The following important conclusions were made from the experiments and the simulations: (i) external arginine additionally supplied to the medium, (ii) oxygen limitation, and (iii) absence of PHA accumulation exerted positive effects on CGP accumulation. These conclusions were utilized to obtain CGP contents in the cells of as high as 17.9% (w x w(-1)) during cultivation of the investigated bacteria at the 30-L scale using mineral salts medium. Such high CGP contents were previously not obtained with these bacteria at a 30-L scale, even if complex media were used.

Application of a KDPG-aldolase gene-dependent addiction system for enhanced production of cyanophycin in Ralstonia eutropha strain H16.

Two different recombinant plasmids both containing the cyanophycin synthetase gene (cphA) of Synechocystis sp. strain PCC6308 but differing concerning the resistance marker gene were tested for their suitability to produce high amounts of cyanophycin in recombinant strains of Ralstonia eutropha. Various cultivation experiments at the 30-L scale revealed very low cyanophycin contents of the cells ranging from 4.6% to 6.2% (w/w) of cellular dry weight (CDW) only, most probably because most cells had lost the corresponding plasmid during cultivation. To establish a cost effective and high efficient system for production of cyanophycin at larger scales using recombinant strains of R. eutropha, we applied two strategies: First, we integrated cphA into the dispensable chromosomal l-lactate dehydrogenase gene (ldh) of R. eutropha. Depending on the cultivation conditions used, relatively low cyanophycin contents between 2.2% and 7.7% (w/w) of CDW were reproducibly detected, which might be due to weak expression or low gene dosage in the single cphA copy strain of R. eutropha. In a second strategy we constructed a KDPG-aldolase gene (eda)-dependent addiction system, which combined features of a multi-copy plasmid with stabilized expression of cphA. Flasks experiments revealed that the cells accumulated extraordinarily high amounts of cyanophycin between 26.9% and 40.0% (w/w) of CDW even under cultivation conditions lacking cyanophycin precursor substrates or plasmid stabilizing antibiotics. Cyanophycin contents of up to 40.0% (w/w) of CDW were also obtained at a 30-L scale or a 500-L pilot-plant scale under such non-selective conditions. This demonstrates impressively that the stabilizing effect of the constructed eda-dependent addiction system can be used for production of enhanced amounts of cyanophycin at a larger scale in recombinant strains of R. eutropha.

Identification of the Anabaena sp. strain PCC7120 cyanophycin synthetase as suitable enzyme for production of cyanophycin in gram-negative bacteria like Pseudomonas putida and Ralstonia eutropha.

The cyanophycin synthetase gene cphA1 encoding the major cyanophycin synthetase (CphA) of Anabaena sp. strain PCC7120 was expressed in Escherichia coli conferring so far the highest specific CphA activity to E. coli (6.7 nmol arginine per min and mg protein). CphA1 and cphA genes of Synechocystis sp. strains PCC6803 and PCC6308 and Synechococcus strain MA19 were also expressed in wild types and polyhydroxyalkanoate-negative (PHA) mutants of Pseudomonas putida and Ralstonia eutropha. Recombinant strains of these bacteria expressing cphA1 accumulated generally more cyanophycin (23.0 and 20.0% of cellular dry matter, CDM, respectively) than recombinants expressing any other cphA (6.8, 9.0, or 15.8% of CDM for P. putida strains and 7.3, 12.6, or 14.1% of CDM for R. eutropha). Furthermore, PHA-negative mutants of P. putida (9.7, 10.0, 17.5, or 24.0% of CDM) and R. eutropha (8.9, 13.8, 16.0, or 22.0% of CDM) accumulated generally more cyanophycin than the corresponding PHA-positive parent strains (6.8, 9.0, 15.8, and 23.0% of CDM for P. putida strains and 7.3, 12.6, 14.1, or 20.0% of CDM for R. eutropha strains). Recombinant strains of Gram-positive bacteria (Bacillus megaterium, Corynebacterium glutamicum) were not suitable for cyanophycin production due to accumulation of less cyanophycin and retarded release of cyanophycin. PHA-negative mutants of P. putida and R. eutropha expressing cphA1 of Anabaena sp. strain PCC7120 are therefore preferred candidates for industrial production of cyanophycin.