The amino acid sequence of the extracellular domain of the virus-encoded M2 matrix protein (peptide M2e) is conserved among all subtypes of influenza A strains, enabling the development of a broad-range vaccine against them. We expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005 (H5N1) in nuclear-transformed duckweed plants for further development of an avian influenza vaccine. The 30-amino acid N-terminal fragment of M2, including M2e (denoted M130), was selected for expression. The M2e DNA sequence fused in-frame to the 3′ end of ricin toxin B chain (RTB) was cloned under control of the CaMV 35S promoter into pBI121. The resulting plasmid was used for duckweed transformation, and 23 independent transgenic duckweed lines were obtained. Asialofetuin-binding ELISA of protein samples from the transgenic plants using polyclonal anti-RTB antibodies confirmed the expression of the RTB–M130 fusion protein in 20 lines. Quantitative ELISA of crude protein extracts from these lines showed RTB–M130 accumulation ranging from 0.25–2.5 μg/g fresh weight (0.0006–0.01% of total soluble protein). Affinity chromatography with immobilized asialofetuin and western blot analysis of protein samples from the transgenic plants showed expression of fusion protein RTB–M130 in the aggregate form with a molecular mass of about 70 kDa. Mice were immunized orally with a preparation of total soluble protein from transgenic plants, receiving four doses of 7 μg duckweed-derived RTB–M130 each, with no additional adjuvant. Specific IgG against M2e was detected in immunized mice, and the endpoint titer of nti-M2e IgG was 1,024. It was confirmed that oral immunization with RTB-M130 induces production of specific antibodies against peptide M2e, one of the most conserved antigens of the influenza virus. These results may provide further information for the development of a duckweed-based expression system to produce a broad-range edible vaccine against avian influenza.

Plant tissue is composed of many different types of cells. Plant cells required to withstand mechanical pressure, such as vessel elements and fibers, have a secondary cell wall consisting of polysaccharides and lignin, which strengthen the cell wall structure and stabilize the cell shape. Previous attempts to alter the properties of the cell wall have mainly focused on reducing the amount of lignin or altering its structure in order to ease its extraction from raw woody materials for the pulp and paper and biorefinery industries. In this work, we propose the in vivo modification of the cell wall structure and mechanical properties by the introduction of resilin, an elastic protein that is able to crosslink with lignin monomers during cell wall synthesis. The effects of resilin were studied in transgenic eucalyptus plants. The protein was detected within the cell wall and its expression led to an increase in the elastic modulus of transgenic stems. In addition, transgenic stems displayed a higher yield point and toughness, indicating that they were able to absorb more energy before breaking.

A series of four cellulose nanocrystal (CNC) suspensions were prepared from bleached softwood kraft pulp using different conditions of sulfuric acid hydrolysis. The CNCs were identical in size (95 nm in length × 5 nm in width) but had different surface charges corresponding to the harshness of the hydrolysis conditions. Consequently, it was possible to isolate the effects of surface charge on the self-assembly and viscosity of the CNC suspensions across surface charges ranging from 0.27%S to 0.89%S. The four suspensions (never-dried, free of added electrolyte) all underwent liquid crystalline phase separation, but the concentration onset for the emergence of the chiral nematic phase shifted to higher values with increasing surface charge. Similarly, suspension viscosity was also influenced by surface charge, with suspensions of lower surface charge CNCs more viscous and tending to gel at lower concentrations. The properties of the suspensions were interpreted in terms of the increase in effective diameter of the nanocrystals due to the surface electrostatic repulsion of the negative sulfate half-esters, as modified by the screening effects of the H+ counterions in the suspensions. The results suggest that there is a threshold surface charge density (∼0.3%S) above which effective volume considerations are dominant across the concentration range relevant to liquid crystalline phase formation. Above this threshold value, phase separation occurs at the same effective volume fraction of CNCs (∼10 vol %), with a corresponding increase in critical concentration due to the decrease in effective diameter that occurs with increasing surface charge. Below or near this threshold value, the formation of end-to-end aggregates may favor gelation and interfere with ordered phase formation.A series of four cellulose nanocrystal (CNC) suspensions were prepared from bleached softwood kraft pulp using different conditions of sulfuric acid hydrolysis. The CNCs were identical in size (95 nm in length × 5 nm in width) but had different surface charges corresponding to the harshness of the hydrolysis conditions. Consequently, it was possible to isolate the effects of surface charge on the self-assembly and viscosity of the CNC suspensions across surface charges ranging from 0.27%S to 0.89%S. The four suspensions (never-dried, free of added electrolyte) all underwent liquid crystalline phase separation, but the concentration onset for the emergence of the chiral nematic phase shifted to higher values with increasing surface charge. Similarly, suspension viscosity was also influenced by surface charge, with suspensions of lower surface charge CNCs more viscous and tending to gel at lower concentrations. The properties of the suspensions were interpreted in terms of the increase in effective diameter of the nanocrystals due to the surface electrostatic repulsion of the negative sulfate half-esters, as modified by the screening effects of the H+ counterions in the suspensions. The results suggest that there is a threshold surface charge density (∼0.3%S) above which effective volume considerations are dominant across the concentration range relevant to liquid crystalline phase formation. Above this threshold value, phase separation occurs at the same effective volume fraction of CNCs (∼10 vol %), with a corresponding increase in critical concentration due to the decrease in effective diameter that occurs with increasing surface charge. Below or near this threshold value, the formation of end-to-end aggregates may favor gelation and interfere with ordered phase formation.

Abstract The demonstration of reliable and stable white light-emitting diodes (LEDs) is one of the main technological challenges of the LED industry. This is usually accomplished by incorporation of light-emitting rare-earth elements (REEs) compounds within an external polymeric coating of a blue LED allowing the generation of white light. However, due to both environmental and cost issues, the development of low-cost REE-free coatings, which exhibit competitive performance compared to conventional white LED is of great importance. In this work, the formation of an REE-free white LED coating is demonstrated. This biocomposite material, composed of biological (crystalline nanocellulose and porcine gastric mucin) and organic (light-emitting dyes) compounds, exhibits excellent optical and mechanical properties as well as resistance to heat, humidity, and UV radiation. The coating is further used to demonstrate a working white LED by incorporating it within a commercial blue LED.

Controlling the permeability and porosity of an inorganic layer using biomolecule building blocks has raised interest for nanotechnological applications. The challenge lies mostly in the fabrication, usually a long, expensive and tedious process, involving many steps. Using biomaterials for this purpose is highly appealing; due to both ease of fabrication and the final output, that contains a bioelement. The biomolecule, specifically, stable protein 1 (SP1), serving as the scaffold for our pattern, is of great stability and durability, and presents size, charge and structural selectivity towards electroactive species. Here, we demonstrate the ability of SP1 to form a rigid template within a sol-gel matrix, allowing selective electron transfer to the gold electrode. Specifically, a thiolated SP1 was first adsorbed on a gold surface followed by filling the non-occupied areas by sol-gel. The latter was electrochemically deposited. The various steps were carefully characterized. Finally, we studied the electrochemistry of numerous redox couple at the Au/SP1/sol-gel interface and found that the nanochannel array shows charge and structural selectivity, which is based on the interactions between the redox species and the functionalities of SP1. The resulted surface shows promise towards electrochemical sensing applications.

Abstract The importance of symbiotic dinitrogen (N2) fixation in shaping the coupled nitrogen–carbon cycle is now known for most humid terrestrial ecosystems. However, whether N2 fixation can play a key role in the nitrogen and carbon budget of water-limited and seasonally dry ecosystems remains a mystery. The maintenance of metabolically and physiologically costly symbiotic fixation in water-limited environments is highly complex. These costs are particularly high during the first developmental season, when allocation to deep rooting and drought resistance mechanisms is essential for seedling survival of prolonged seasonal drought. We, therefore, evaluated how drought-adapted legume species change their allocation to symbiotic nitrogen fixation as a function of soil nitrogen availability. We tested this on seedlings of a suite of four common Mediterranean legume shrubs with a strong seasonal behaviour, which we grew under controlled nitrogen and phosphorus availabilities. We asked: (1) Do species differ in their investment and regulation of nitrogen fixation? (2) Is fixation regulated via plant allocation to nodules, fixation rate or both? and (3) Does phosphorus availability limit symbiotic nitrogen fixation? All Mediterranean perennial legumes in the experiment established and grew, nodulated, and fixed nitrogen, even under severe nitrogen limitation. The four species reacted similarly to nitrogen supply, by strongly downregulating fixation through both decreased nodulation and lower rate of fixation. However, we found a significant interspecific difference in fixation (both nodulation and rate), biomass production and growth rate. Our experimental species presented a range of fixation investment strategies corresponding to life-history and resource partitioning patterns. Phosphorus limitation had a minor influence on both fixation and plant growth. Synthesis. The high physiological cost of symbiotic fixation imposes the need to tightly regulate fixation in perennial legumes coping with severe water stress. Control of fixation allows legume species to colonize recently disturbed nitrogen-deficient habitats, cope with grazing, survive long seasonal droughts and recover nitrogen fixation later in the wet season, and survive over time by reducing nitrogen inputs to the ecosystem.

The cosmopolitan coccolithophore Emiliania huxleyi is a unicellular eukaryotic alga that forms vast blooms in the oceans impacting large biogeochemical cycles. These blooms are often terminated due to infection by the large dsDNA virus, E. huxleyi virus (EhV). It was recently established that EhV-induced modulation of E. huxleyi metabolism is a key factor for optimal viral infection cycle. Despite the huge ecological importance of this host–virus interaction, the ability to assess its spatial and temporal dynamics and its possible impact on nutrient fluxes is limited by current approaches that focus on quantification of viral abundance and biodiversity. Here, we applied a host and virus gene expression analysis as a sensitive tool to quantify the dynamics of this interaction during a natural E. huxleyi bloom in the North Atlantic. We used viral gene expression profiling as an index for the level of active infection and showed that the latter correlated with water column depth. Intriguingly, this suggests a possible sinking mechanism for removing infected cells as aggregates from the E. huxleyi population in the surface layer into deeper waters. Viral infection was also highly correlated with induction of host metabolic genes involved in host life cycle, sphingolipid, and antioxidant metabolism, providing evidence for modulation of host metabolism under natural conditions. The ability to track and quantify defined phases of infection by monitoring co-expression of viral and host genes, coupled with advance omics approaches, will enable a deeper understanding of the impact that viruses have on the environment.

Diatoms are one of the key phytoplankton groups in the ocean, forming vast oceanic blooms and playing a significant part in global primary production. To shed light on the role of redox metabolism in diatom's acclimation to light–dark transition and its interplay with cell fate regulation, we generated transgenic lines of the diatom Thalassiosira pseudonana that express the redox-sensitive green fluorescent protein targeted to various subcellular organelles. We detected organelle-specific redox patterns in response to oxidative stress, indicating compartmentalized antioxidant capacities. Monitoring the GSH redox potential (EGSH) in the chloroplast over diurnal cycles revealed distinct rhythmic patterns. Intriguingly, in the dark, cells exhibited reduced basal chloroplast EGSH but higher sensitivity to oxidative stress than cells in the light. This dark-dependent sensitivity to oxidative stress was a result of a depleted pool of reduced glutathione which accumulated during the light period. Interestingly, reduction in the chloroplast EGSH was observed in the light phase prior to the transition to darkness, suggesting an anticipatory phase. Rapid chloroplast EGSH re-oxidation was observed upon re-illumination, signifying an induction of an oxidative signaling during transition to light that may regulate downstream metabolic processes. Since light–dark transitions can dictate metabolic capabilities and susceptibility to a range of environmental stress conditions, deepening our understanding of the molecular components mediating the light-dependent redox signals may provide novel insights into cell fate regulation and its impact on oceanic bloom successions.

The biotroph wheat powdery mildew, Blumeria graminis (DC.) E.O. Speer, f. sp. tritici Em. Marchal (Bgt), has undergone long and dynamic co-evolution with its hosts. In the last 10,000 years, processes involved in plant evolution under domestication, altered host-population structure. Recently both virulence and genomic profiling separated Bgt into two groups based on their origin from domestic host and from wild emmer wheat. While most studies focused on the Bgt pathogen, there is significant knowledge gaps in the role of wheat host diversity in this specification. This study aimed to fill this gap by exploring qualitatively and also quantitatively the disease response of diverse host panel to powdery mildew [105 domesticated wheat genotypes (Triticum turgidum ssp. dicoccum, T. turgidum ssp. durum, and T. aestivum) and 241 accessions of its direct progenitor, wild emmer wheat (T. turgidum ssp. dicoccoides)]. A set of eight Bgt isolates, originally collected from domesticated and wild wheat was used for screening this wheat collection. The isolates from domesticated wheat elicited susceptible to moderate plant responses on domesticated wheat lines and high resistance on wild genotypes (51.7% of the tested lines were resistant). Isolates from wild emmer elicited reciprocal disease responses: high resistance of domesticated germplasm and high susceptibility of the wild material (their original host). Analysis of variance of the quantitative phenotypic responses showed a significant Isolates × Host species interaction [P(F) < 0.0001] and further supported these findings. Furthermore, analysis of the range of disease severity values showed that when the group of host genotypes was inoculated with Bgt isolate from the reciprocal host, coefficient of variation was significantly higher than when inoculated with its own isolates. This trend was attributed to the role of major resistance genes in the latter scenario (high proportion of complete resistance). By testing the association between disease severity and geographical distance from the source of inoculum, we have found higher susceptibility in wild emmer close to the source. Both qualitative and quantitative assays showed a reciprocal resistance pattern in the wheat host and are well aligned with the recent findings of significant differentiation into wild-emmer and domesticated-wheat populations in the pathogen.

{Drought is the major environmental factor limiting wheat production worldwide. Developing novel cultivars with greater drought tolerance is the most viable solution to ensure sustainable agricultural production and alleviating threats to food-security. Here we established a core-collection of landraces and modern durum wheat cultivars (WheatME

Abstract The molecular mechanism regulating dormancy release in grapevine buds is as yet unclear. It was formerly proposed that dormancy is maintained by abscisic acid (ABA)-mediated repression of bud–meristem activity and that removal of this repression triggers dormancy release. It was also proposed that such removal of repression may be achieved via natural or artificial up-regulation of VvA8H-CYP707A4, which encodes ABA 8′-hydroxylase, and is the most highly expressed paralog in grapevine buds. The current study further examines these assumptions, and its experiments reveal that (a) hypoxia and ethylene, stimuli of bud dormancy release, enhance expression of VvA8H-CYP707A4 within grape buds, (b) the VvA8H-CYP707A4 protein accumulates during the natural transition to the dormancy release stage, and (c) transgenic vines overexpressing VvA8H-CYP707A4 exhibit increased ABA catabolism and significant enhancement of bud break in controlled and natural environments and longer basal summer laterals. The results suggest that VvA8H-CYP707A4 functions as an ABA degrading enzyme, and are consistent with a model in which the VvA8H-CYP707A4 level in the bud is up-regulated by natural and artificial bud break stimuli, which leads to increased ABA degradation capacity, removal of endogenous ABA-mediated repression, and enhanced regrowth. Interestingly, it also hints at sharing of regulatory steps between latent and lateral bud outgrowth.

Pre-visual detection of crop disease is critical for food security. Field-based spectroscopic remote sensing offers a method to enable timely detection, but still requires appropriate instrumentation and testing. Soybean plants were spectrally measured throughout a growing season to assess the capacity of leaf and canopy level spectral measurements to detect non-visual foliage symptoms induced by Fusarium virguliforme (Fv, which causes sudden death syndrome). Canopy reflectance measurements were made using the Piccolo Doppio dual field-of-view, two-spectrometer (400 to 1630 nm) system on a tractor. Leaf level measurements were obtained, in different plots, using a handheld spectrometer (400 to 2500 nm). Partial least squares discriminant analysis (PLSDA) was applied to the spectroscopic data to discriminate between Fv-inoculated and control plants. Canopy and leaf spectral data allowed identification of Fv infection, prior to visual symptoms, with classification accuracy of 88% and 91% for calibration, 79% and 87% for cross-validation, and 82% and 92% for validation, respectively. Differences in wavelengths important to prediction by canopy vs. leaf data confirm that there are different bases for accurate predictions among methods. Partial least square regression (PLSR) was used on a late-stage canopy level data to predict soybean seed yield, with calibration, cross-validation and validation R2 values 0.71, 0.59 and 0.62 (p < 0.01), respectively, and validation root mean square error of 0.31 t·ha−1. Spectral data from the tractor mounted system are thus sensitive to the expression of Fv root infection at canopy scale prior to canopy symptoms, suggesting such systems may be effective for precision agricultural research and management.

This study investigated the impact of canopy cover and seasonality on litter decay in Mediterranean pine forests to enhance climate predictions.

Abstract Decomposition of organic matter in semi-arid ecosystems is a key component of the terrestrial carbon (C) cycle. The well-known inaccuracies in predicting litter decay in water-limited regions were lessened by considering solar radiation as an abiotic decay driver of photodegradation. Moreover, exposure to high solar irradiance in dry periods often led to massive facilitation of litter decay in subsequent wet periods (“photoacceleration”), though in many studies this effect was absent. Recently, water vapour and dew were identified as modulators enabling substantial microbial degradation during rainless periods. Here, we investigated, (1) whether the activity of micro-organisms modifies litter traits, such as litter quality and microbial community in dry periods, consequently altering the loss of litter mass and nitrogen (N) in wet periods, and (2) whether it can co-occur with photoacceleration. By successively introducing litter to the field at the beginning and the end of the dry season, we found that microbial activity during the dry season affected litter mass and N loss during the wet season. Low microbial activity in the dry season led to inhibition of mass loss in the wet season, while high microbial activity led to facilitation of mass loss. Microbial activity during the dry season also caused strong inhibition of N loss from litter during the wet season, likely by enhancing the dry-season N loss. A microclimate manipulation experiment using radiation filters showed that microbial activity and exposure to solar radiation jointly modified the litter during the dry season and affected subsequent decay in the wet season. Knowledge of biotic and abiotic modifications of litter during dry periods and their implication for wet periods enhances our understanding of litter decay in semi-arid regions. Furthermore, it can improve biogeochemical model predictions of C and N cycling in drylands and in the many regions that are projected to experience a drier climate during the coming decades. A plain language summary is available for this article.

Plant litter decomposition in drylands is not well understood, and even less is known about decay of the abundant standing dead residues. Here, we followed decomposition of standing and surface litter, and assessed the underlying drivers and mechanisms.

Spontaneous Raman scattering microspectroscopy, second harmonic generation (SHG) and 2-photon excited fluorescence (2PF) were used in combination to characterize the morphology together with the chemical composition of the cell wall in native plant tissues. As the data obtained with unstained sections of Sorghum bicolor root and leaf tissues illustrate, nonresonant as well as pre-resonant Raman microscopy in combination with hyperspectral analysis reveals details about the distribution and composition of the major cell wall constituents. Multivariate analysis of the Raman data allows separation of different tissue regions, specifically the endodermis, xylem and lumen. The orientation of cellulose microfibrils is obtained from polarization-resolved SHG signals. Furthermore, 2-photon autofluorescence images can be used to image lignification. The combined compositional, morphological and orientational information in the proposed coupling of SHG, Raman imaging and 2PF presents an extension of existing vibrational microspectroscopic imaging and multiphoton microscopic approaches not only for plant tissues.

Plants do not have mineralized skeletons. Instead, each of the plant's cells has an envelope of a cellulose-based wall, which provides a mechanical support to the organism. This stiff wall enables plants to assume flexible body shapes. However, the wall interferes with proteinous muscle-like movements of cells and organs because it is too stiff to yield to forces generated by motor proteins. Nevertheless, plants move constantly. The movements rely on water translocations, which result in the swelling (or growth) of cells located strategically. Water may swell protoplasts in movements that require live cells, like tip growth, tropism, and gas exchange. Other movements are initiated by the swelling of cell walls. These occur in dead tissues that can afford drying. The hygroscopically based movement is very common in seed dispersal mechanisms. The seed that detaches from the mother plant is carried by a cellulosic device. This device was synthesized by the plant and programmed to do some mechanical work, like jumping, crawling, and sowing, in order to deliver the seed to a germination location. This nonliving device provides the seed with means to move away from its mother and siblings. The movement may utilize several types of cells, which differ in the arrangement of cell wall cellulose microfibrils. I present here three types of contracting cells that, together with stiff fiber cells resisting any contraction, create a variety of hygroscopic movements.

The awn in stork’s bill (Erodium gruinum) seed dispersal units coils as it dries. This hygroscopic movement promotes the dissemination and sowing of the seeds. Here we aimed to understand the movement rate, by correlating water dynamics within the awn to the spatial variation in the chemical composition of the awn’s cell walls. We followed the hygroscopic movement visually and measured the kinetics of water adsorption–desorption in segments along the awn. We integrated data from white light, fluorescence, and Raman microscopy, and Matrix Assisted Laser Desorption Ionization imaging to characterize the micro chemical makeup of the awn. We hydrolyzed awns and followed the change in the cell walls’ composition and the effect on the movement. We found that the coil’s top segment is more sensitive to humidity changes than the coil’s base. At the top part of the coil, we found high concentration of modified lignin. In comparison, the base part of the awn contained lower concentration of mostly unmodified lignin. Ferulic acid concentration increased along the awn, apparently cross-linking hemicellulose and strengthening cell-to-cell adhesion. We propose that the high concentration of modified lignin at the coil’s top increased the hydrophobicity of the cell walls, allowed faster water molecules dynamics; thus inducing fast reaction to ambient humidity. Strong cell-to-cell adhesion in this region created a durable tissue required for the awn’s repeated movement that is induced by the diurnal humidity cycles.