Leveraging substrate flexibility and product selectivity of acetogens in two-stage systems for chemical production

Carbon dioxide (CO₂) stands out as sustainable feedstock for developing a circular carbon economy whose energy supply could be obtained by boosting the production of clean hydrogen from renewable electricity. H₂-dependent CO₂ gas fermentation using acetogenic microorganisms offers a viable solution of increasingly demonstrated value. While gas fermentation advances to achieve commercial process scalability, which is currently limited to a few products such as acetate and ethanol, it is worth taking the best of the current state-of-the-art technology by its integration within innovative bioconversion schemes. This review presents multiple scenarios where gas fermentation by acetogens integrate into double-stage biotechnological production processes that use CO₂ as sole carbon feedstock and H₂ as energy carrier for products' synthesis. In the integration schemes here reviewed, the first stage can be biotic or abiotic while the second stage is biotic. When the first stage is biotic, acetogens act as a biological platform to generate chemical intermediates such as acetate, formate and ethanol that become substrates for a second fermentation stage. This approach holds the potential to enhance process titre/rate/yield metrics and products' spectrum. Alternatively, when the first stage is abiotic, the integrated two-stage scheme foresees, in the first stage, the catalytic transformation of CO₂ into C₁ products that, in the second stage, can be metabolized by acetogens. This latter scheme leverages the metabolic flexibility of acetogens in efficient utilization of the products of CO₂ abiotic hydrogenation, namely formate and methanol, to synthesize multicarbon compounds but also to act as flexible catalysts for hydrogen storage or production.     

Direct identification of the rotary angle of ATP cleavage in F1-ATPase from Bacillus PS3

F₁-ATPase is the world’s smallest biological rotary motor driven by ATP hydrolysis at three catalytic β subunits. The 120° rotational step of the central shaft γ consists of 80° substep driven by ATP binding and a subsequent 40° substep. In order to correlate timing of ATP cleavage at a specific catalytic site with a rotary angle, we designed a new F₁-ATPase (F₁) from thermophilic Bacillus PS3 carrying β(E190D/F414E/F420E) mutations, which cause extremely slow rates of both ATP cleavage and ATP binding. We produced an F₁ molecule that consists of one mutant β and two wild-type βs (hybrid F₁). As a result, the new hybrid F₁ showed two pausing angles that are separated by 200°. They are attributable to two slowed reaction steps in the mutated β, thus providing the direct evidence that ATP cleavage occurs at 200° rather than 80° subsequent to ATP binding at 0°. This scenario resolves the long-standing unclarified issue in the chemomechanical coupling scheme and gives insights into the mechanism of driving unidirectional rotation.     

Physiological mechanism of drought tolerance in transgenic rice plants expressing Capsicum annuum methionine sulfoxide reductase B2 (CaMsrB2) gene

The methionine sulfoxide reductase B2 (MsrB2) gene catalyzes the reduction of free and protein-bound methionine sulfoxide to methionine and is known to provide tolerance to biotic and abiotic environmental stresses. There are yet to be any reports that MsrB2 enhances drought tolerance. Two drought-tolerant transgenic rice lines, L-8 (single copy) and L-23 (two copy), expressing the Capsicum annuum MsrB2 (CaMsrB2) gene were selected for stress tolerance phenotyping under drought stress conditions. CaMsrB2 enhanced relative water content, maintained substantial quantum yield (F _v /F _m ratio), and subsequently improved photosynthetic pigments. Interestingly, L-23, carrying two-copy T-DNA insertion, showed greater drought tolerance through more effective stomatal regulation, carotenoid concentration, and osmotic potential than the wild type. High-tech infrared technology (FLIR SC620) was used for the selection of stress-tolerant physiotypes. Later, the IR results were correlated with other tested physiological parameters. The IR images, average plant temperature, and physiological parameters of the treated plants are discussed in detail.     

Biological production of H2, CH4 and CO2 in the deep subsurface of the Iberian Pyrite Belt

Most of the terrestrial deep subsurfaces are oligotrophic environments in which some gases, mainly H₂, CH₄ and CO₂, play an important role as energy and/or carbon sources. In this work, we assessed their biotic and abiotic origin in samples from subsurface hard-rock cores of the Iberian Pyrite Belt (IPB) at three different depths (414, 497 and 520 m). One set of samples was sterilized (abiotic control) and all samples were incubated under anaerobic conditions. Our results showed that H₂, CH₄ and CO₂ remained low and constant in the sterilized controls while their levels were 4, 4.1 and 2.5 times higher respectively, in the unsterilized samples compared to the abiotic controls. The δ¹³C_CH4-values measured in the samples (range −31.2 to −43.0 ‰) reveals carbon isotopic signatures that are within the range for biological methane production. Possible microorganisms responsible for the biotic production of the gases were assessed by CARD-FISH. The analysis of sequenced genomes of detected microorganisms within the subsurface of the IPB allowed to identify possible metabolic activities involved in H₂ (Rhodoplanes, Shewanella and Desulfosporosinus), CH₄ (Methanobacteriales) and CO₂ production. The obtained results suggest that part of the H₂, CH₄ and CO₂ detected in the deep subsurface has a biological origin.     

Transgenic insect-resistant maintainer line (IR68899B) for improvement of hybrid rice

Heterosis has helped to increase rice yield in F₁ hybrids by 15–20% beyond the level of inbred semidwarf varieties. For stable yield performance rice hybrids must also possess genetic resistance to biotic stresses. One of these, stem borer, reduces the expected yield of hybrid rice. The truncated synthetic cryIA(b) gene from Bacillus thuringiensis is known to be effective in controlling stem borer. The development of transformation techniques has provided the technology for incorporating this bacterial gene into the rice genome, which has not been possible by conventional breeding methods. We have introduced a new approach of using a transgenic maintainer line for developing an insect-resistant hybrid rice. An elite IRRI maintainer line (IR68899B) has been transformed with the cryIA(b) gene driven by the 35S constitutive promoter using the biolistic method. The integration and expression of the cryIA(b) gene could be demonstrated through Southern and Western blot analyses that have been carried out so far up to the T₂ generations. Insect bioassay data showed an enhanced resistance to yellow stem borer in the Bt ⁺ transgenic plants. This is the first report of the development of a transgenic maintainer line for use in hybrid rice improvement. Received: 17 December 1997 / Revision received: 23 June 1998 / Accepted: 25 September 1998     

Interactions Among Abiotic Drivers,Disturbance and Gross Ecosystem Carbon Exchange on Soil Respiration from Subtropical Pine Savannas

Globally, soil CO₂ efflux rates (F_s) have been linked to changes in soil water content (SWC), rainfall and temperature and/or productivity. However, within an ecosystem, F_s can vary based on site structure and function, which can be affected by a combination of abiotic and biotic factors. This becomes particularly important when an ecosystem is faced with disturbances, such as drought or fire. Site-specific compensatory responses to disturbances may therefore alter C mineralization, as well as root respiration. Hence, single location F_s estimates may not be a representative for ecosystems across their distributional ranges. We conducted a 6-year study along an edaphic moisture gradient of longleaf pine ecosystems that were maintained with prescribed fire, using eddy covariance and soil respiration measurements to address how F_s varies with changes in ecosystem structure and function, as well as disturbances. Lower air temperatures (T_air) decreased F_s at all sites, but that response was also affected by productivity and SWC. Productivity significantly altered F_s rates at all sites, especially when we accounted for changes in temperature and SWC. Plant regrowth post-fire temporarily increased F_s (10–40%), whereas drought reduced F_s at all sites. Our results show that site productivity, F_s and the degree to which ecosystems adapt to climate variations and disturbance can be site specific. Hence, model forecasting of carbon dynamics would strongly benefit from multi-location measurements of F_s across the distributional range of an ecosystem.     

Identification of biotic and abiotic stress up-regulated ESTs in Gossypium arboreum

Asiatic desi cotton (Gossypium arboreum) shows great potential against biotic and abiotic stresses. The stress resistant nature makes it a best source for the identification of biotic and abiotic stress resistant genes. As in many plants same set of genes show responding behavior against the various abiotic and biotic stresses. Thus in the present study the ESTs from the G. arboreum drought stressed leaves were subjected to find the up-regulated ESTs in abiotic and biotic stresses through homology and in-silico analysis. A cDNA library has been constructed from the drought stressed G. arboreum plant. 778 clones were randomly picked and sequenced. All these sequences were subjected to in-silico identification of biotic and abiotic up-regulated ESTs. Total 39 abiotic and biotic up-regulated ESTs were identified. The results were further validated by real-time PCR; by randomly selection of ten ESTs. These findings will help to develop stress resistant crop varieties for better yield and growth performance under stresses.     

Transgenic approaches for genetic improvement in groundnut (Arachis hypogaea L.) against major biotic and abiotic stress factors

Cultivated groundnut (Arachis hypogaea L.) is considered as one of the primary oilseed crops and a major fodder for cattle industry in most of the developing countries, owing to its rich source of protein. It is due to its geocarpic nature of growth that the overall yield performance of groundnut is hindered by several biotic and abiotic stress factors. Multidimensional attempts were undertaken to combat these factors by developing superior groundnut varieties, modified with integral mechanism of tolerance/resistance; however this approach proved to be futile, owing to inferior pod and kernel quality. As a superior alternative, biotechnological intervention like transformation of foreign genes, either directly (biolistic) or via Agrobacterium, significantly aided in the development of advanced groundnut genotypes equipped with integral resistance against stresses and enhanced yield attributing traits. Several genes triggered by biotic and abiotic stresses, were detected and some of them were cloned and transformed as major parts of transgenic programmes. Application of modern molecular biological techniques, in designing biotic and abiotic stress tolerant/resistant groundnut varieties that exhibited mechanisms of resistance, relied on the expression of specific genes associated to particular stress. The genetically transformed stress tolerant groundnut varieties possess the potential to be employed as donor parents in traditional breeding programmes for developing varieties that are resilient to fungal, bacterial, and viral diseases, as well as to draught and salinity. The present review emphasizes on the retrospect and prospect of genetic transformation tools, implemented for the enhancement of groundnut varieties against key biotic and abiotic stress factors.     

Genomic and chemical evidence for local adaptation in resistance to different herbivores in Datura stramonium

Because most species are collections of genetically variable populations distributed to habitats differing in their abiotic/biotic environmental factors and community composition, the pattern and strength of natural selection imposed by species on each other's traits are also expected to be highly spatially variable. Here, we used genomic and quantitative genetic approaches to understand how spatially variable selection operates on the genetic basis of plant defenses to herbivores. To this end, an F₂ progeny was generated by crossing Datura stramonium (Solanaceae) parents from two populations differing in their level of chemical defense. This F₂ progeny was reciprocally transplanted into the parental plants’ habitats and by measuring the identity by descent (IBD) relationship of each F₂ plant to each parent, we were able to elucidate how spatially variable selection imposed by herbivores operated on the genetic background (IBD) of resistance to herbivory, promoting local adaptation. The results highlight that plants possessing the highest total alkaloid concentrations (sum of all alkaloid classes) were not the most well-defended or fit. Instead, specific alkaloids and their linked loci/alleles were favored by selection imposed by different herbivores. This has led to population differentiation in plant defenses and thus, to local adaptation driven by plant-herbivore interactions.     

Abiotic stress and transgenics: Implications for reproductive success and crop-to-wild gene flow in Brassicas

Various abiotic and biotic stressors affect crop and weed plant performance in agroecosystems. Ozone (O₃) tolerance in plants is partly regulated by the genotype and phenotypical properties, and it varies greatly in related species of wild and crop backgrounds. Thus, a continuous increase in atmospheric O₃ concentration could change population dynamics of sexually compatible crop and weed species, and consequently affect crop-to-wild gene flow in the future. One way to build resistance against a biotic stressor, in this case insect-mediated herbivory, in crop plants is transgene-mediated insecticidal toxin production. In this study we aimed to describe how the physiological and phenological responses in a crop Brassica and its weedy relatives functioned to affect their comparative O₃ tolerance. Furthermore, we studied how harbouring a transgene affects these responses in B. napus and B. rapa × transgenic B. napus BC₂F₂ backcross hybrid plants to reveal any within-plant trade-offs among toxin production, growth and O₃ tolerance. We found a higher number of O₃ symptoms but more effective compensatory assimilate allocation directed to reproduction for wild B. rapa compared to crop B. napus under elevated O₃. This result suggested that the invasion-orientated strategy of producing a high number of seeds when vegetative growth is limited might improve the performance of weedy species under elevated O₃. The probabilities for crop-to-wild transgene flow could be increased through higher seed production in hybrids under elevated O₃, but the germination of hybrid seeds in particular was hampered by O₃. The presence of transgenes did not perturb fecundity, within-plant biomass allocation or O₃ tolerance of B. napus.     

A review of the allozyme data set for the Canarian endemic flora: causes of the high genetic diversity levels and implications for conservation

Background and Aims

Allozyme and reproductive data sets for the Canarian flora are updated in order to assess how the present levels and structuring of genetic variation have been influenced by the abiotic island traits and by phylogenetically determined biotic traits of the corresponding taxa; and in order to suggest conservation guidelines.

Methods

Kruskal–Wallis tests are conducted to assess the relationships of 27 variables with genetic diversity (estimated by A, P, H_o and H_e) and structuring (G_ST) of 123 taxa representing 309 populations and 16 families. Multiple linear regression analyses (MLRAs) are carried out to determine the relative influence of the less correlated significant abiotic and biotic factors on the genetic diversity levels.

Key Results and Conclusions

The interactions between biotic features of the colonizing taxa and the abiotic island features drive plant diversification in the Canarian flora. However, the lower weight of closeness to the mainland than of (respectively) high basic chromosome number, partial or total self-incompatibility and polyploidy in the MLRAs indicates substantial phylogenetic constraint; the importance of a high chromosome number is feasibly due to the generation of a larger number of linkage groups, which increase gametic and genotypic diversity. Genetic structure is also more influenced by biotic factors (long-range seed dispersal, basic chromosome number and partial or total self-incompatibility) than by distance to the mainland. Conservation-wise, genetic structure estimates (F_ST/G_ST) only reflect endangerment under intensive population sampling designs, and neutral genetic variation levels do not directly relate to threat status or to small population sizes. Habitat protection is emphasized, but the results suggest the need for urgent implementation of elementary reproductive studies in all cases, and for ex situ conservation measures for the most endangered taxa, even without prior studies. In non-endangered endemics, multidisciplinary research is needed before suggesting case-specific conservation strategies. The molecular information relevant for conservation should be conserved in a standardized format to facilitate further insight.     

Complementation of the Fo c Subunit of Escherichia coli with That of Streptococcus mutans and Properties of the Hybrid FoF1 ATP Synthase

The c subunit of Streptococcus mutans ATP synthase (F_oF₁) is functionally exchangeable with that of Escherichia coli, since E. coli with a hybrid F_oF₁ is able to grow on minimum succinate medium through oxidative phosphorylation. E. coli F₁ bound to the hybrid F_o with the S. mutans c subunit showed N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity similar to that of E. coli F_oF₁. Thus, the S. mutans c subunit assembled into a functional F_o together with the E. coli a and b subunits, forming a normal F₁ binding site. Although the H⁺ pathway should be functional, as was suggested by the growth on minimum succinate medium, ATP-driven H⁺ transport could not be detected with inverted membrane vesicles in vitro. This observation is partly explained by the presence of an acidic residue (Glu-20) in the first transmembrane helix of the S. mutans c subunit, since the site-directed mutant carrying Gln-20 partly recovered the ATP-driven H⁺ transport. Since S. mutans is recognized to be a primary etiological agent of human dental caries and is one cause of bacterial endocarditis, our system that expresses hybrid F_o with the S. mutans c subunit would be helpful to find antibiotics and chemicals specifically directed to S. mutans.     

Molecular mapping of a gene ‘ld(t)’ controlling cleistogamy in rice

Cleistogamy is the self-pollination within closed spikelets and is expected to be a useful genetic tool for prevention of possible gene transfer in transgenic crops, for maintenance of genetic purity in autogamous crops, and for increased tolerance to biotic and abiotic stresses. Mapping of the gene ld(t), which is responsible for lack of lodicules inside spikelets and causes cleistogamy, was carried out using F₂ and F₃ populations derived from a cleistogamous (CL) mutant CL-SNU × Milyang 23 cross. A number of STS markers along chromosomes were developed and bulked segregant analysis was adopted for preliminary mapping. The results showed that the ld(t) was located at the end region of chromosome 1L, flanked by S01178b (an STS marker developed for the locus at 178 cM based on the rice genetic map reported by Japanese Rice Genome Project) at 0.8 cM and co-segregated with S01181a and S01181b (an STS marker developed for the locus at 181 cM).     

Visualization of Secale cereale DNA in wheat germ plasm by fluorescent in situ hybridization

Homozygous wheat/rye (1BL/1RS or 1AS/ 1RL) translocation lines have significantly contributed to wheat production, and several other wheat/rye translocation lines show a potential promise against biotic and abiotic stresses. Detecting the presence of rye at the chromosome level is feasible by C-banding and isozyme protocols, but the diagnostic strength of genomic in situ hybridization for eventually analyzing smaller DNA introgressions has greater significance. As a first step we have applied the genomic in situ hybridization technique to detect rye chromosomes in a wheat background using germ plasm of agricultural significance. By this method rye contributions to the translocations 1BL/1RS, 1AL/1RS, 5AS/5RL and 6BS/6RL could be identified. Differential labelling has further enabled the detection of rye and Thinopyrum bessarabicum chromosomes in a trigeneric hybrid of Triticum aestivum/Th. bessarabicum//Secale cereale.     

Interspecific Hybridization of Pennisetum glaucum (L) R. Br. with Wild Relatives

To widen the germplasm base for the introgression of economically important traits such as resistance to biotic and abiotic stresses from related species, crosses of cultivated pearl millet were made with pollen from four related species differing in the basic chromosome number (x=5,7,8 and 9). Embryo rescue technique was used to obtain viable progeny. Pollinations of pearl millet with Pennisetum ramosum (2n=2x=10) did not give any viable progeny. Pearl millet interspecific hybrids with P. schwelnfurthii (2n-2x-14), P. mezianum (2n=4x-32) and P. orientale (2n=2x=18) were obtained. The hybrid between P. glaucum and P. mezianum (2n=23) is the first successful report. Interspecific hybrid plants resembled their corresponding pollen parents. Southern blots of Psfl digested DNAs from interspecific hybrids and the parental species were hybridized to a full length rDNA to further confirm their hybridity. This further revealed differential amplification of two rDNA repeats among the F₁ hybrids from the same cross (P. glaucum X P. orientale).     

Transgressive segregation of primary and secondary metabolites in F<Subscript>2</Subscript> hybrids between <Emphasis Type="Italic">Jacobaea aquatica</Emphasis> and <Emphasis Type="Italic">J. vulgaris</Emphasis>

Hybridization between plant species can have a number of biological consequences; interspecific hybridization has been tied to speciation events, biological invasions, and diversification at the level of genes, metabolites, and phenotypes. This study aims to provide evidence of transgressive segregation in the expression of primary and secondary metabolites in hybrids between Jacobaea vulgaris and J. aquaticus using an NMR-based metabolomic profiling approach. A number of F₂ hybrid genotypes exhibited metabolomic profiles that were outside the range encompassed by parental species. Expression of a number of primary and secondary metabolites, including jacaronone analogues, chlorogenic acid, sucrose, glucose, malic acid, and two amino acids was extreme in some F₂ hybrid genotypes compared to parental genotypes, and citric acid was expressed in highest concentrations in J. vulgaris. Metabolomic profiling based on NMR is a useful tool for quantifying genetically controlled differences between major primary and secondary metabolites among plant genotypes. Interspecific plant hybrids in general, and specifically hybrids between J. vulgaris and J. aquatica, will be useful for disentangling the ecological role of suites of primary and secondary metabolites in plants, because interspecific hybridization generates extreme metabolomic diversity compared to that normally observed between parental genotypes.