Lignin engineering in field-grown poplar trees affects the endosphere bacterial microbiome

Cinnamoyl-CoA reductase (CCR), an enzyme central to the lignin bio-synthetic pathway, represents a promising biotechnological target to reduce lignin levels and to improve the commercial viability of lignocellulosic biomass. However, silencing of the CCR gene results in considerable flux changes of the general and monolignol-specific lignin pathways, ultimately leading to the accumulation of various extractable phenolic compounds in the xylem. Here, we evaluated host genotype-dependent effects of field-grown, CCR-down-regulated poplar trees (Populus tremula x Populus alba) on the bacterial rhizosphere microbiome and the endosphere microbiome, namely the microbiota present in roots, stems, and leaves. Plant-associated bacteria were isolated from all plant compartments by selective isolation and enrichment techniques with specific phenolic carbon sources (such as ferulic acid) that are up-regulated in CCR-deficient poplar trees. The bacterial microbiomes present in the endosphere were highly responsive to the CCR-deficient poplar genotype with remarkably different metabolic capacities and associated community structures compared with the WT trees. In contrast, the rhizosphere microbiome of CCR-deficient and WT poplar trees featured highly overlapping bacterial community structures and metabolic capacities. We demonstrate the host genotype modulation of the plant microbiome by minute genetic variations in the plant genome. Hence, these interactions need to be taken into consideration to understand the full consequences of plant metabolic pathway engineering and its relation with the environment and the intended genetic improvement

M & L Jaargang 11/3

GeneriekPatrick Vissers en Michiel Heirman Het Sint-Fredeganduskerkhof te Deurne. Is er leven na de dood? [The Saint Fredegand graveyard in Deurne (Antwerp). Is there any life after death?]Ooit de vredigste rustplaats van menig welstellend Antwerpenaar zag het kerkhof rond de Sint-Fredeganduskerk te Deurne méér dan lijfelijk de dood voor ogen. Afgeschreven, dan weer beschermd maar openlijk ten prooi gelaten aan vernieling, leek het einde daarnet nog onomkeerbaar aangebroken. Toch volstaat voor Patrick Vissers en Michiel Heirman één oogopslag m de fascinatie te ondergaan van dit groots, zwaar met herinneringen beladen park.Roland Op De Beeck De gebeeldhouwde retabels: een typisch produkt van het Antwerpse kunstambacht (1420-1560). [Sculptured retabels. A typical produce of the Antwerp artistic crafts (1420-1560).]Niet zelden monumentaal, uitgevoerd in de beste materialen en door de handigste ambachtslui, vormden de 16de-eeuwse Antwerpse retabels een gewaarmerkt en fel gegeerd handelsproduct. Van het Iberisch schiereiland tot de Baltische zee, getuigt dit oorspronkelijk onmisbaar cultusobject nog steeds van een ooit artistiek hoogstaande én winstgevende Vlaamse activiteit. Vooruitlopend op een ambitieuze restauratiecampagne, laat Roland Op De Beeck ons kennis maken met deze gouden wonderen.Lode De Clercq De restauratie van het Ghémarmonument op het kerkhof te Laken. [The restoration of the Ghemar mausoleum in Laeken (Brussels).]Begenadigde oorden als het kerkhof van Laken vormen ware openlucht-schouwplaatsen voor klein-architectuur en monumentaal beeldhouwwerk. Wetenschappers vinden er dan weer hun gading als onderzoeksterrein voor de weinig bezoedelde gedragingen van gesteenten. Zèlf nauw betrokken bij de restauratie van het mausoleum voor de familie Ghémar, maakt Lode De Clercq duidelijk in welke mate het grafmonument al deze eigenschappen combineert en derhalve diende te worden gered.SummaryM&L Binnenkran

Performance of 16s rDNA Primer Pairs in the Study of Rhizosphere and Endosphere Bacterial Microbiomes in Metabarcoding Studies

Next-generation sequencing technologies have revolutionized the methods for studying microbial ecology by enabling high resolutioncommunity profiling. However, the use of these technologies in unraveling the plant microbiome remains challenging. Many bacterial 16S rDNA primer pairs also exhibit high affinity for non-target DNA such as plastid (mostly chloroplast) DNA and mitochondrial DNA. Therefore, we experimentally tested a series of commonly used primers for the analysis of plant associated bacterial communities using 454 pyrosequencing. We evaluated the performance of all selected primer pairs in the study of the bacterial microbiomes present in the rhizosphere soil, root, stem and leaf endosphere of field-grown poplar trees (Populus tremula x Populus alba) based on (a) co-amplification of non-target DNA, (b) low amplification efficiency for pure chloroplast DNA (real-time PCR), (c) high retrieval of bacterial 16S rDNA, (d) high operational taxonomic unit (OTU) richness and Inverse Simpson diversity and (e) taxonomic assignment of reads. Results indicate that experimental evaluation of primers provide valuable information that could contribute in the selection of suitable primer pairs for 16S rDNA metabarcoding studies in plant-microbiota research. Furthermore, we show that primer pair 799F-1391R outperforms all other primer pairs in our study in the elimination of non target DNA and retrieval of bacterial OTUs

Direct catalytic conversion of cellulose to liquid straight-chain alkanes

High yields of liquid straight-chain alkanes were obtained directly from cellulosic feedstock in a one-pot biphasic catalytic system. The catalytic reaction proceeds at elevated temperatures under hydrogen pressure in the presence of tungstosilicic acid, dissolved in the aqueous phase, and modified Ru/C, suspended in the organic phase. Tungstosilicic acid is primarily responsible for cellulose hydrolysis and dehydration steps, while the modified Ru/C selectively hydrogenates intermediates en route to the liquid alkanes. Under optimal conditions, microcrystalline cellulose is converted to 82% n-decane-soluble products, mainly n-hexane, within a few hours, with a minimum formation of gaseous and char products. The dominant route to the liquid alkanes proceeds via 5-hydroxymethylfurfural (HMF), whereas the more common pathway via sorbitol appears to be less efficient. High liquid alkane yields were possible through (i) selective conversion of cellulose to glucose and further to HMF by gradually heating the reactor, (ii) a proper hydrothermal modification of commercial Ru/C to tune its chemoselectivity to furan hydrogenation rather than glucose hydrogenation, and (iii) the use of a biphasic reaction system with optimal partitioning of the intermediates and catalytic reactions. The catalytic system is capable of converting subsequent batches of fresh cellulose, enabling accumulation of the liquid alkanes in the organic phase during subsequent runs. Its robustness is illustrated in the conversion of the raw (soft)wood sawdust

Direct catalytic conversion of cellulose to liquid straight-chain alkanes

High yields of liquid straight-chain alkanes were obtained directly from cellulosic feedstock in a one-pot biphasic catalytic system. The catalytic reaction proceeds at elevated temperatures under hydrogen pressure in the presence of tungstosilicic acid, dissolved in the aqueous phase, and modified Ru/C, suspended in the organic phase. Tungstosilicic acid is primarily responsible for cellulose hydrolysis and dehydration steps, while the modified Ru/C selectively hydrogenates intermediates en route to the liquid alkanes. Under optimal conditions, microcrystalline cellulose is converted to 82% n-decane-soluble products, mainly n-hexane, within a few hours, with a minimum formation of gaseous and char products. The dominant route to the liquid alkanes proceeds via 5-hydroxymethylfurfural (HMF), whereas the more common pathway via sorbitol appears to be less efficient. High liquid alkane yields were possible through (i) selective conversion of cellulose to glucose and further to HMF by gradually heating the reactor, (ii) a proper hydrothermal modification of commercial Ru/C to tune its chemoselectivity to furan hydrogenation rather than glucose hydrogenation, and (iii) the use of a biphasic reaction system with optimal partitioning of the intermediates and catalytic reactions. The catalytic system is capable of converting subsequent batches of fresh cellulose, enabling accumulation of the liquid alkanes in the organic phase during subsequent runs. Its robustness is illustrated in the conversion of the raw (soft)wood sawdust

Effects of topping and non-topping on growth-regulating hormones of flue-cured tobacco (Nicotiana tabacum L.)—a proteomic analysis

IntroductionUntil now, the mechanism underlying the impact of topping on hormone regulation in tobacco plants remains unclear, and most studies investigating the hormone signaling pathways in plants rely on genes or transcriptional pathways.MethodsThis study examines the regulatory mechanisms of hormones in the roots and leaves of tobacco plants with and without topping at the protein level.ResultsThe results demonstrate that, compared with non-topped plants, topping leads to a decrease in the levels of IAA (auxin), ABA (abscisic acid), and GA (gibberellin) hormones in the leaves, whereas the content of the JA (jasmonic acid) hormone increases. Furthermore, in the roots, topping results in an increase in the levels of IAA, ABA, and JA hormones, along with a decrease in GA content. In the leaves, a total of 258 significantly different proteins were identified before and after topping, with 128 proteins upregulated and 130 proteins downregulated. In the roots, there were 439 proteins with significantly different quantities before and after topping, consisting of 211 upregulated proteins and 228 downregulated proteins. Notably, these proteins were closely associated with the metabolic and biosynthetic pathways of secondary metabolites, as indicated by functional categorization.ConclusionsWhen integrating the hormone changes and the proteomics results, it is evident that topping leads to increased metabolic activity and enhanced hormone synthesis in the root system. This research provides a theoretical foundation for further investigations into the regulation and signaling mechanisms of hormones at the protein level before and after topping in plants

Fungal extracellular polymeric substance matrices – Highly specialized microenvironments that allow fungi to control soil organic matter decomposition reactions

Filamentous fungi play a key role in the terrestrial carbon cycle as they are the primary decomposers of lignocellulose in soil organic matter (SOM). Fungi secrete a wide range of oxidative and hydrolytic enzymes, and generate radicals through extracellular secondary metabolites to decompose SOM. To study fungal decomposition of SOM, the activities of isolated enzymes are typically studied as proxies for the decomposition activity of fungi. However, extracellular enzymes involved in lignocellulose decomposition are often bound to fungal extracellular polymeric substance (EPS) matrices. This association between extracellular enzymes and EPS matrices affects the activities of the enzymes. Moreover, extracellular enzymes and fungal cells are prone to attack by radicals and proteolytic enzymes themselves. Hence, these seemingly incompatible decomposition mechanisms must be regulated in some way in the fungal extracellular space to allow efficient decomposition of SOM, while preventing damage to secreted extracellular enzymes or the fungal cells themselves. We here review studies investigating the associations between fungal extracellular enzymes and EPS matrices and how these associations affect hydrolytic and oxidative reactions involved in SOM decomposition. Based on the knowledge compiled in the current review, we propose that fungal EPS matrices should be viewed as highly dynamic and functional parts of the fungal extracellular decomposition machinery. We also build a conceptual illustration that describes how the molecular composition and structure of fungal EPS matrices ensure that extracellular decomposition reactions only proceed at the right time and in the right place

Decomposition of soil organic matter by ectomycorrhizal fungi : Mechanisms and consequences for organic nitrogen uptake and soil carbon stabilization

A major fraction of nitrogen (N) in boreal forest soils is found in organic forms associated with soil organic matter (SOM) and mineral particles. The capacity of ectomycorrhizal (ECM) fungal symbionts to access this N is debated, considering that these fungi have lost many of the genes for decomposing organic matter that were present in their saprotrophic ancestors. To gain a molecular-level understanding of the N-mining processes in ECM fungi, we developed an experimental approach where the processes of decomposition were studied in parallel with the changes in the structure and properties of the organic matter. We showed that ECM fungi have significant capacities to assimilate organic N associated with SOM and mineral surfaces. The decomposition mechanisms differ between species, reflecting the lignocellulose decomposition mechanisms found in their saprotrophic ancestors. During N-mining, the ECM fungi processed the SOM to a material with increased adsorptive properties to iron oxide mineral particles. Two pathways contributed to these changes: Extracellular modifications of the SOM and secretion of mineral surface reactive metabolites. Some of these metabolites have iron(III)-reducing activities and can participate in extracellular Fenton reactions and redox reactions at iron oxide mineral surfaces. We conclude that the traditional framework for understanding organic N acquisition by ECM fungi from recalcitrant SOM must be extended to a framework that includes how those decomposition activities affect the stabilization and reactivity of mineral-associated SOM. The activity through these complex networks of reactions is decisive for the overall effect of ECM fungal decomposition on nutrients and C-cycling in forest ecosystems

Ectomycorrhizal Fungal Transformation of Dissolved Organic Matter : Consequences for Reductive Iron Oxide Dissolution and Fenton-Based Oxidation of Mineral-Associated Organic Matter

Recent studies have shown that dissolved organic matter (DOM) decomposed by ectomycorrhizal (ECM) fungi increases adsorptive properties of organic matter towards soil mineral surfaces. Concomitantly, ECM fungi secrete secondary metabolites with iron reducing capacity that are thought to participate in non-enzymatic Fenton-based decomposition of DOM. The aim of this study was to investigate if the iron reduction induced by the ECM fungus Paxillus involutus during organic matter decomposition was conserved in the decomposed DOM. We explored how the modified DOM reductively dissolved ferrihydrite and goethite nanoparticles and how these processes affected the reactions with H2O2 and the Fenton-based oxidation of mineral-associated organic matter. Culture filtrates were obtained from incubation of the ECM fungus on DOM from forest litter of a spruce forest. This modified DOM was separated by extraction into an ethyl acetate and a water fraction. These fractions were reacted with ferrihydrite and goethite in absence and presence of H2O2. Dissolved Fe2+ and HO• were measured and the reactions at the iron oxide-water interfaces were monitored in real-time with in-situ IR spectroscopy. Experiments showed that decomposition of DOM by P. involutus generated a modified DOM that displayed an increased and persistent reductive capacity. Most of the reductants were isolated in the aromatic- and carboxyl-dominated ethyl acetate fraction but some reduction capacity was also captured in the water fraction mainly containing carbohydrates. Reductive dissolution was more extensive for ferrihydrite than goethite, and this process generated significant oxidation of the DOM-ferrihydrite associations. Oxidation of adsorbed DOM was triggered by H2O2 via heterogenous and homogeneous Fenton reactions. These oxidation processes were favored by ferrihydrite because of a high reduction potential and a high efficiency of heterogeneous Fenton as compared to goethite. An optimal timing between the heterogeneous and homogeneous Fenton processes triggered extensive radical oxidation of the DOM-ferrihydrite associations generating a high concentration of surface-associated oxalate. Overall, the results show that organic matter associated with ferrihydrite may be more susceptible to radical oxidation than on goethite, and that fungal decomposition of DOM in general may have consequences for other important soil processes such as mineral dissolution, adsorption and initiation of radical reactions

OCTAVVS : A graphical toolbox for high-throughput preprocessing and analysis of vibrational spectroscopy imaging data

Modern vibrational spectroscopy techniques enable the rapid collection of thousands of spectra in a single hyperspectral image, allowing researchers to study spatially heterogeneous samples at micrometer resolution. A number of algorithms have been developed to correct for effects such as atmospheric absorption, light scattering by cellular structures and varying baseline levels. After preprocessing, spectra are commonly decomposed and clustered to reveal informative patterns and subtle spectral changes. Several of these steps are slow, labor-intensive and require programming skills to make use of published algorithms and code. We here present a free and platform-independent graphical toolbox that allows rapid preprocessing of large sets of spectroscopic images, including atmospheric correction and a new algorithm for resonant Mie scattering with improved speed. The software also includes modules for decomposition into constituent spectra using the popular Multivariate Curve Resolution–Alternating Least Squares (MCR-ALS) algorithm, augmented by region-of-interest selection, as well as clustering and cluster annotation