Silicon alleviates antimony phytotoxicity in giant reed (Arundo donax L.).

MAIN CONCLUSION: Silicon enhances photosynthetic efficiency, biomass, and lignification of root structures possibly limiting antimony translocation and mitigating phytotoxicity in giant reed plants. Antimony (Sb) is a non-essential metalloid causing toxic effects in plants. Silicon has been reported to impart tolerance against biotic and abiotic stress in plants. Fast-growing plant, giant reed (Arundo donax L.) is a promising energy crop, can be a suitable plant for phytoremediation. However, information regarding the tolerance capacity with respect to Sb toxicity and potential of Si to mitigate the Sb phytotoxicity in giant reed are very scarce. Rhizomes of giant reed were grown for ten weeks in hydroponics exposed to Sb, Si, and their combination wherein treatment without Sb/Si served as control. Effect of these treatments on rate of net photosynthesis and photosynthetic pigments, phytoextraction ability of Sb, Si and Sb uptake, plant biomass, and lignification and suberization of roots along with localization of Sb and Si were analysed. We found that Si considerably improved the growth and biomass of giant reed under Sb toxicity. Antimony reduced the photosynthesis and decreased the content of photosynthetic pigments, which was completely alleviated by Si. Silicon amendment to Sb treated plants enhanced root lignification. Silicon enhanced lignification of root structures probably restricted the Sb translocation. However, co-localization of Sb with Si has not been observed neither at the shoot nor at the root levels. Similarly, Sb was also not detected in leaf phytoliths. These findings suggest that Si treatment promotes overall plant growth by improving photosynthetic parameters and decreasing Sb translocation from root to shoot in giant reed by improving root lignification.

Recently photoassimilated carbon and fungus-delivered nitrogen are spatially correlated in the ectomycorrhizal tissue of Fagus sylvatica.

Ectomycorrhizal plants trade plant-assimilated carbon for soil nutrients with their fungal partners. The underlying mechanisms, however, are not fully understood. Here we investigate the exchange of carbon for nitrogen in the ectomycorrhizal symbiosis of Fagus sylvatica across different spatial scales from the root system to the cellular level. We provided 15 N-labelled nitrogen to mycorrhizal hyphae associated with one half of the root system of young beech trees, while exposing plants to a 13 CO2 atmosphere. We analysed the short-term distribution of 13 C and 15 N in the root system with isotope-ratio mass spectrometry, and at the cellular scale within a mycorrhizal root tip with nanoscale secondary ion mass spectrometry (NanoSIMS). At the root system scale, plants did not allocate more 13 C to root parts that received more 15 N. Nanoscale secondary ion mass spectrometry imaging, however, revealed a highly heterogenous, and spatially significantly correlated distribution of 13 C and 15 N at the cellular scale. Our results indicate that, on a coarse scale, plants do not allocate a larger proportion of photoassimilated C to root parts associated with N-delivering ectomycorrhizal fungi. Within the ectomycorrhizal tissue, however, recently plant-assimilated C and fungus-delivered N were spatially strongly coupled. Here, NanoSIMS visualisation provides an initial insight into the regulation of ectomycorrhizal C and N exchange at the microscale.

A new insight on structural and some functional aspects of peri-endodermal thickenings, a specific layer in Noccaea caerulescens roots.

BACKGROUND AND AIMS: Cell walls of the peri-endodermis, a layer adjacent to the endodermis in alpine pennycress (Noccaea caerulescens) roots, form C-shaped peri-endodermal thickenings (PETs). Despite its specific position close to the endodermis, the assumed similarity of PETs to phi thickenings in many other species, and the fact that N. caerulescens is a well-studied heavy-metal-hyperaccumulating plant, the PET as a root trait is still not understood. METHODS: Here, we characterized PET cell walls by histochemical techniques, Raman spectroscopy, immunolabelling and electron microscopy. Moreover, a role of PETs in solute transport was tested and compared with Arabidopsis thaliana plants, which do not form PETs in roots. KEY RESULTS: Cell walls with PETs have a structured relief mainly composed of cellulose and lignin. Suberin, typical of endodermal cells, is missing but pectins are present on the inner surface of the PET. Penetrating dyes are not able to cross PETs either by the apoplasmic or the symplasmic pathway, and a significantly higher content of metals is found in root tissues outside of PETs than in innermost tissues. CONCLUSIONS: Based on their development and chemical composition, PETs are different from the endodermis and closely resemble phi thickenings. Contrarily, the different structure and dye impermeability of PETs, not known in the case of phi thickenings, point to an additional barrier function which makes the peri-endodermis with PETs a unique and rare layer.

Response of Arabidopsis halleri to cesium and strontium in hydroponics: Extraction potential and effects on morphology and physiology.

Stable isotopes of cesium (Cs) and strontium (Sr) as well as their radioactive isotopes are of serious environmental concern. The pollution of the biosphere, particularly soil and water has received considerable attention for removal of these contaminants in recent years. Arabidopsis halleri (A. halleri) is a hyperaccumulator plant species able to take up large amounts of several metals into its above ground organs without showing significant signs of toxicity. Therefore, we investigated responses, metal accumulation and element distribution in roots and leaves of A. halleri after treatment with stable Cs and Sr. Plants were hydroponically grown in different concentrations of cesium sulfate (between 0.002 and 20 mM) and strontium nitrate (between 0.001 and 100 mM). Uptake of Cs and Sr into leaves was analyzed from extracts by inductively coupled plasma mass spectrometry (ICP-MS). Although internal concentration of Cs and Sr increased with rising external concentrations, the amount of accumulated metal in relation to available metal decreased. Therefore, the potential of the plant to effectively transfer metals from growth medium to leaves occurred at low and moderate concentrations, whereas after that when the concentration of metal increased further the transfer factors were decreased. A. halleri accumulated Sr more efficiently than Cs. The transfer factors were higher for Sr (up to 184) than for Cs (up to 16). The results indicate positive correlation of Cs and Sr accumulation to K and Ca transport to leaves. The toxicity of Cs and Sr was assessed by measuring photosynthetic efficiency and growth parameters. In leaves, Cs and Sr affected the chlorophyll fluorescence at their low and high concentrations. Significant reduction of plant growth (dry weight of roots and leaves) was observed at Sr concentrations >0.01 mM. Cs-treated plants exhibited only decreased length of leaves at concentrations>0.02 mM. The distribution of the elements within the different tissues of leaves and roots was investigated by using Energy Dispersive X-Ray microanalysis (EDX) with a scanning electron microscope (SEM). EDX revealed that Cs and Sr were accumulated differently in root and leaf tissues. The hydroponic experiment showed a potential for A. halleri to treat hotspots with radioactive Cs and Sr.

Adaptive responses of mature giant chloroplasts in the deep-shade lycopod Selaginella erythropus to prolonged light and dark periods.

Deep-shade plants have adapted to low-light conditions by varying morphology and physiology of cells and chloroplasts, but it still remains unclear, if prolonged periods of high-light or darkness induce additional modifications in chloroplasts' anatomy and pigment patterns. We studied giant chloroplasts (bizonoplasts) of the deep-shade lycopod Selaginella erythropus in epidermal cells of mature fully developed microphylls and subjected them to prolonged darkness and high-light conditions. Chloroplast size and ultrastructure were investigated by light and electron microscopy. Physiological traits were studied by pigment analyses, photosynthetic performance of photosystem II, and formation of reactive oxygen species. Results show that (a) thylakoid patterns and shape of mature bizonoplasts vary in response to light and dark conditions. (b) Prolonged darkness induces transitory formation of prolamellar bodies, which so far have not been described in mature chloroplasts. (c) Photosynthetic activity is linked to structural responses of chloroplasts. (d) Photosystem II is less active in the upper zone of bizonoplasts and more efficient in the grana region. (e) Formation of reactive oxygen species reflects the stress level caused by high-light. We conclude that during prolonged darkness, chlorophyll persists and even increases; prolamellar bodies form de novo in mature chloroplasts; bizonoplasts have spatial heterogeneity of photosynthetic performance.

Is the Binding Pattern of Zinc(II) Equal in Different Bryophyte Species?

Bryophytes are usually taken as good bioindicators. However, they represent a large group of terrestrial plants and they express an enormous range of peculiarities within the plant kingdom. With the aim to search for a common pattern of zinc binding, we established axenical in vitro cultures of a dozen bryophyte species that include hornworts, thallose, and leafy liverworts, as well as acrocarp and pleurocarp mosses. The species were grown free of contaminants for many years prior to the application of different treatments, i.e. offering Zn(II) from solid and liquid media and in combination with different anions. The localization and binding of zinc was detected by confocal microscopy using the zinc-specific dye FluoZin™-3. In one of the species, Hypnum cupressiforme (which is widely used for atmospheric heavy metal deposition studies in biomonitoring), semi-quantitative analyses of zinc were performed by energy dispersive X-ray microspectrometry (EDX) in a scanning electron microscope. The results suggest no common pattern of Zn(II) binding in different bryophyte species. Instead, the binding pattern seems to be species specific. Zinc is located in certain areas or cellular compartments, as clearly shown by the EDX measurements in H. cupressiforme.

Investigation of Calcium Forms in Lichens from Travertine Sites.

Lichens are symbiotic organisms with an extraordinary capability to colonise areas of extreme climate and heavily contaminated sites, such as metal-rich habitats. Lichens have developed several mechanisms to overcome the toxicity of metals, including the ability to bind metal cations to extracellular sites of symbiotic partners and to subsequently form oxalates. Calcium is an essential alkaline earth element that is important in various cell processes. Calcium can serve as a metal ligand but can be toxic at elevated concentrations. This study investigated calcium-rich and calcium-poor sites and the lichen species that inhabit them (Cladonia sp.). The calcium content of these lichen species were analyzed, along with localized calcium oxalate formed in thalli collected from each site. The highest concentration of calcium was found in the lichen squamules, which can serve as a final deposit for detoxification. Interestingly, the highest content of calcium in Cladonia furcata was localized to the upper part of the thallus, which is the youngest. The produced calcium oxalates were species-specific. Whewellite (CaC2O4∙H2O) was formed in the case of C. furcata and weddellite (CaC2O4∙2H2O) was identified in C. foliacea.

Both abundant and rare fungi colonizing Fagus sylvatica ectomycorrhizal root-tips shape associated bacterial communities.

Ectomycorrhizal fungi live in close association with their host plants and form complex interactions with bacterial/archaeal communities in soil. We investigated whether abundant or rare ectomycorrhizal fungi on root-tips of young beech trees (Fagus sylvatica) shape bacterial/archaeal communities. We sequenced 16S rRNA genes and fungal internal transcribed spacer regions of individual root-tips and used ecological networks to detect the tendency of certain assemblies of fungal and bacterial/archaeal taxa to inhabit the same root-tip (i.e. modularity). Individual ectomycorrhizal root-tips hosted distinct fungal communities associated with unique bacterial/archaeal communities. The structure of the fungal-bacterial/archaeal association was determined by both, dominant and rare fungi. Integrating our data in a conceptual framework suggests that the effect of rare fungi on the bacterial/archaeal communities of ectomycorrhizal root-tips contributes to assemblages of bacteria/archaea on root-tips. This highlights the potential impact of complex fine-scale interactions between root-tip associated fungi and other soil microorganisms for the ectomycorrhizal symbiosis.

The response of the accumulator plants Noccaea caerulescens, Noccaea goesingense and Plantago major towards the uranium.

Uranium (U) is a naturally occurring metal; its environmental levels can be increased due to processes in the nuclear industry and fertilizer production. The transfer of U in the food chain from plants is associated with deleterious chemical and radiation effects. To date, limited information is available about U toxicity on plant physiology. This study investigates the responses of metal-accumulating plants to different concentrations of U. The plants Noccaea caerulescens and Noccaea goesingense are known as metal hyperaccumulators and therefore could serve as candidates for the phytoremediation of radioactive hotspots; Plantago major is a widely used pharmaceutical plant that pioneers polluted grounds and therefore should not contain high concentrations of toxic elements. The experimental plants were grown hydroponically at U concentrations between 1 µM and 10 mM. The content of U and essential elements was analyzed in roots and leaves by ICP-MS. The amount of accumulated U was influenced by its concentration in the hydroponics. Roots contained most of the metal, whereas less was transported up to the leaves, with the exception of N. goesingense in a medium concentration of U. U also influenced the nutrient profile of the plants. We localized the U in plant tissues using EDX in the SEM. U was evenly distributed in roots and leaves of Noccaea species, with one exception in the roots of N. goesingense, where the central cylinder contained more U than the cortex. The toxicity of U was assessed by measuring growth and photosynthetic parameters. While root biomass of N. caerulescens was not affected by U, root biomass of N. goesingense decreased significantly at high U concentrations of 0.1 and 10 mM and root biomass of P. major decreased at 10 mM U. Dry weight of leaves was decreased at different U concentrations in the three plant species; a promotive effect was observed in N. caerulescens at lowest concentration offered. Chlorophyll a fluorescence was not affected or negatively affected by U in both Noccaea species, whereas in Plantago also positive effects were observed. Our results show that the impact of U on Plantago and Noccaea relates to its external concentration and to the plant species. When growing in contaminated areas, P. major should not be used for medicinal purpose. Noccaea species and P. major could immobilize U in their rhizosphere in hotspots contaminated by U, and they could extract limited amounts of U into their leaves.

Gland cell responses to feeding in Drosera capensis, a carnivorous plant.

Glands of Drosera absorb and transport nutrients from captured prey, but the mechanism and dynamics remain unclear. In this study, we offered animal proteins in the form of fluorescent albumin (FITC-BSA) and observed the reactions of the glands by live cell imaging and fluorescence microscopy. The ultrastructure of these highly dynamic processes was also assessed in high-pressure frozen and freeze substituted (HPF-FS) cells. HPF-FS yielded excellent preservation of the cytoplasm of all cell types, although the cytosol looked different in gland cells as compared to endodermoid and stalk cells. Especially prominent were the ER and its contacts with the plasma membrane, plasmodesmata, and other organelles as well as continuities between organelles. Also distinct were actin microfilaments in association with ER and organelles. Application of FITC-BSA to glands caused the formation of fluorescent endosomes that pinched off the plasma membrane. Endosomes fused to larger aggregates, and accumulated in the bulk cytoplasm around the nucleus. They did not fuse with the cell sap vacuole but remained for at least three days; in addition, fluorescent vesicles also proceeded through endodermoid and transfer cells to the epidermal and parenchymal cells of the tentacle stalk.

Metal accumulation in the acrocarp moss Atrichum undulatum under controlled conditions.

Mosses are frequently used to monitor atmospheric metal contamination but few studies on metal adsorption under controlled conditions are available. Here, the accumulation of the heavy metals copper and zinc was studied in the acrocarp moss Atrichum undulatum. An in vitro culture of A. undulatum was established and the same line, size and equally old remets were exposed to six different treatments representing various metal exposure times and washing scenarios as rain simulation. The metal treatments were done in copper and zinc salts (Cu-acetate, CuSO4, ZnSO4 and ZnCl2, respectively). Energy-Dispersive X-ray microanalysis (EDX) was employed to detect bound heavy metals on the moss plantlets. Element distribution in stems and leaves was measured separately. The aqueous solution of metal salts facilitated an adsorption of both elements in the moss tissue as compared to solid medium. Furthermore, A. undulatum can tolerate pollution of zinc and copper in a distinctive extent; our data point towards a higher zinc tolerance whereas copper is rather harmful. However, semi-quantitatively, less zinc was detected within the moss tissue compared to copper. Interestingly, a strong positive correlation between the accumulation of copper/zinc and iron, and a strong negative correlation between copper/zinc and magnesium, respectively, was documented.

Protein sorting into protein bodies during barley endosperm development is putatively regulated by cytoskeleton members, MVBs and the HvSNF7s.

Cereal endosperm is a short-lived tissue adapted for nutrient storage, containing specialized organelles, such as protein bodies (PBs) and protein storage vacuoles (PSVs), for the accumulation of storage proteins. During development, protein trafficking and storage require an extensive reorganization of the endomembrane system. Consequently, endomembrane-modifying proteins will influence the final grain quality and yield. However, little is known about the molecular mechanism underlying endomembrane system remodeling during barley grain development. By using label-free quantitative proteomics profiling, we quantified 1,822 proteins across developing barley grains. Based on proteome annotation and a homology search, 94 proteins associated with the endomembrane system were identified that exhibited significant changes in abundance during grain development. Clustering analysis allowed characterization of three different development phases; notably, integration of proteomics data with in situ subcellular microscopic analyses showed a high abundance of cytoskeleton proteins associated with acidified PBs at the early development stages. Moreover, endosomal sorting complex required for transport (ESCRT)-related proteins and their transcripts are most abundant at early and mid-development. Specifically, multivesicular bodies (MVBs), and the ESCRT-III HvSNF7 proteins are associated with PBs during barley endosperm development. Together our data identified promising targets to be genetically engineered to modulate seed storage protein accumulation that have a growing role in health and nutritional issues.

Response of Plantago major to cesium and strontium in hydroponics: Absorption and effects on morphology, physiology and photosynthesis.

Human activities lead to increasing concentration of the stable elements cesium (Cs) and strontium (Sr) and their radioactive isotopes in the food chain, where plants play an important part. Here we investigated Plantago major under the influence of long-term exposure to stable Cs and Sr. The plants were cultivated hydroponically in different concentrations of cesium sulfate (between 0.002 and 20 mM) and strontium nitrate (between 0.001 and 100 mM). Uptake of Cs and Sr into leaves was analyzed from extracts by inductively coupled plasma mass spectrometry (ICP-MS). It was increased with increasing external Cs and Sr concentrations. However, the efficiency of Cs and Sr transfer from solution to plants was higher for low external concentrations. Highest transfer factors were 6.78 for Cs and 71.13 for Sr. Accumulation of Sr was accompanied by a slight decrease of potassium (K) and calcium (Ca) in leaves, whereas the presence of Cs in the medium affected only uptake of K. The toxic effects of Cs and Sr were estimated from photosynthetic reactions and plant growth. In leaves, Cs and Sr affected the chlorophyll fluorescence even at their low concentrations. Low and high concentrations of both ions reduced dry weight and length of roots and leaves. The distribution of the elements between the different tissues of leaves and roots was investigated using Energy Dispersive X-Ray microanalysis (EDX) with scanning electron microscope (SEM). Overall, observations suggested differential patterns in accumulating Cs and Sr within the roots and leaves. When present in higher concentrations the amount of Cs and Sr transferred from environment to plants was sufficient to affect some physiological processes. The experimental model showed a potential for P. major to study the influence of radioactive contaminants and their removal from hotspots.

Rapid Transfer of Plant Photosynthates to Soil Bacteria via Ectomycorrhizal Hyphae and Its Interaction With Nitrogen Availability.

Plant roots release recent photosynthates into the rhizosphere, accelerating decomposition of organic matter by saprotrophic soil microbes ("rhizosphere priming effect") which consequently increases nutrient availability for plants. However, about 90% of all higher plant species are mycorrhizal, transferring a significant fraction of their photosynthates directly to their fungal partners. Whether mycorrhizal fungi pass on plant-derived carbon (C) to bacteria in root-distant soil areas, i.e., incite a "hyphosphere priming effect," is not known. Experimental evidence for C transfer from mycorrhizal hyphae to soil bacteria is limited, especially for ectomycorrhizal systems. As ectomycorrhizal fungi possess enzymatic capabilities to degrade organic matter themselves, it remains unclear whether they cooperate with soil bacteria by providing photosynthates, or compete for available nutrients. To investigate a possible C transfer from ectomycorrhizal hyphae to soil bacteria, and its response to changing nutrient availability, we planted young beech trees (Fagus sylvatica) into "split-root" boxes, dividing their root systems into two disconnected soil compartments. Each of these compartments was separated from a litter compartment by a mesh penetrable for fungal hyphae, but not for roots. Plants were exposed to a 13C-CO2-labeled atmosphere, while 15N-labeled ammonium and amino acids were added to one side of the split-root system. We found a rapid transfer of recent photosynthates via ectomycorrhizal hyphae to bacteria in root-distant soil areas. Fungal and bacterial phospholipid fatty acid (PLFA) biomarkers were significantly enriched in hyphae-exclusive compartments 24 h after 13C-CO2-labeling. Isotope imaging with nanometer-scale secondary ion mass spectrometry (NanoSIMS) allowed for the first time in situ visualization of plant-derived C and N taken up by an extraradical fungal hypha, and in microbial cells thriving on hyphal surfaces. When N was added to the litter compartments, bacterial biomass, and the amount of incorporated 13C strongly declined. Interestingly, this effect was also observed in adjacent soil compartments where added N was only available for bacteria through hyphal transport, indicating that ectomycorrhizal fungi were acting on soil bacteria. Together, our results demonstrate that (i) ectomycorrhizal hyphae rapidly transfer plant-derived C to bacterial communities in root-distant areas, and (ii) this transfer promptly responds to changing soil nutrient conditions.

Silicon Uptake and Localisation in Date Palm (Phoenix dactylifera) - A Unique Association With Sclerenchyma.

Date palm (Phoenix dactylifera) can accumulate as much as 1% silicon (Si), but not much is known about the mechanisms inherent to this process. Here, we investigated in detail the uptake, accumulation and distribution of Si in date palms, and the phylogeny of Si transporter genes in plants. We characterized the PdNIP2 transporter following heterologous expression in Xenopus oocytes and used qPCR to determine the relative expression of Si transporter genes. Silicon accumulation and distribution was investigated by light microscopy, scanning electron microscopy coupled with X-ray microanalysis and Raman microspectroscopy. We proved that PdNIP2-1 codes for a functional Si-permeable protein and demonstrated that PdNIP2 transporter genes were constitutively expressed in date palm. Silicon aggregates/phytoliths were found in specific stegmata cells present in roots, stems and leaves and their surfaces were composed of pure silica. Stegmata were organized on the outer surface of the sclerenchyma bundles or associated with the sclerenchyma of the vascular bundles. Phylogenetic analysis clustered NIP2 transporters of the Arecaceae in a sister position to those of the Poaceae. It is suggested, that Si uptake in date palm is mediated by a constitutively expressed Si influx transporter and accumulated as Si aggregates in stegmata cells abundant in the outer surface of the sclerenchyma bundles (fibers).

Using RT-qPCR, Proteomics, and Microscopy to Unravel the Spatio-Temporal Expression and Subcellular Localization of Hordoindolines Across Development in Barley Endosperm.

Hordeum vulgare (barley) hordoindolines (HINs), HINa, HINb1, and HINb2, are orthologous proteins of wheat puroindolines (PINs) that are small, basic, cysteine-rich seed-specific proteins and responsible for grain hardness. Grain hardness is, next to its protein content, a major quality trait. In barley, HINb is most highly expressed in the mid-stage developed endosperm and is associated with both major endosperm texture and grain hardness. However, data required to understand the spatio-temporal dynamics of HIN transcripts and HIN protein regulation during grain filling processes are missing. Using reverse transcription quantitative PCR (RT-qPCR) and proteomics, we analyzed HIN transcript and HIN protein abundance from whole seeds (WSs) at four [6 days after pollination (dap), 10, 12, and ≥20 dap] as well as from aleurone, subaleurone, and starchy endosperm at two (12 and ≥20 dap) developmental stages. At the WS level, results from RT-qPCR, proteomics, and western blot showed a continuous increase of HIN transcript and HIN protein abundance across these four developmental stages. Miroscopic studies revealed HIN localization mainly at the vacuolar membrane in the aleurone, at protein bodies (PBs) in subaleurone and at the periphery of starch granules in the starchy endosperm. Laser microdissetion (LMD) proteomic analyses identified HINb2 as the most prominent HIN protein in starchy endosperm at ≥20 dap. Additionally, our quantification data revealed a poor correlation between transcript and protein levels of HINs in subaleurone during development. Here, we correlated data achieved by RT-qPCR, proteomics, and microscopy that reveal different expression and localization pattern of HINs in each layer during barley endosperm development. This indicates a contribution of each tissue to the regulation of HINs during grain filling. The effect of the high protein abundance of HINs in the starchy endosperm and their localization at the periphery of starch granules at late development stages at the cereal-based end-product quality is discussed. Understanding the spatio-temporal regulated HINs is essential to improve barley quality traits for high end-product quality, as hard texture of the barley grain is regulated by the ratio between HINb/HINa.

Microscopic and Proteomic Analysis of Dissected Developing Barley Endosperm Layers Reveals the Starchy Endosperm as Prominent Storage Tissue for ER-Derived Hordeins Alongside the Accumulation of Barley Protein Disulfide Isomerase (HvPDIL1-1).

Barley (Hordeum vulgare) is one of the major food sources for humans and forage sources for animal livestock. The average grain protein content (GPC) of barley ranges between 8 and 12%. Barley hordeins (i.e., prolamins) account for more than 50% of GPC in mature seeds and are important for both grain and flour quality. Barley endosperm is structured into three distinct cell layers: the starchy endosperm, which acts essentially as storage tissue for starch; the subaleurone, which is characterized by a high accumulation of seed storage proteins (SSPs); and the aleurone, which has a prominent role during seed germination. Prolamins accumulate in distinct, ER-derived protein bodies (PBs) and their trafficking route is spatio-temporally regulated. The protein disulfide isomerase (PDI) has been shown to be involved in PB formation. Here, we unravel the spatio-temporal proteome regulation in barley aleurone, subaleurone, and starchy endosperm for the optimization of end-product quality in barley. We used laser microdissection (LMD) for subsequent nanoLC-MS/MS proteomic analyses in two experiments: in Experiment One, we investigated the proteomes of dissected barley endosperm layers at 12 and at ≥20 days after pollination (DAP). We found a set of 10 proteins that were present in all tissues at both time points. Among these proteins, the relative protein abundance of D-hordein, B3-hordein and HvPDIL1-1 significantly increased in starchy endosperm between 12 and ≥20 DAP, identifying the starchy endosperm as putative major storage tissue. In Experiment Two, we specifically compared the starchy endosperm proteome at 6, 12, and ≥20 DAP. Whereas the relative protein abundance of D-hordein and B3-hordein increased between 6 and ≥20 DAP, HvPDIL1-1 increased between 6 and 12 DAP, but remained constant at ≥20 DAP. Microscopic observations showed that these relative protein abundance alterations were accompanied by additional localization of hordeins at the periphery of starch granules and a partial re-localization of HvPDIL1-1 from PBs to the periphery of starch granules. Our data indicate a spatio-temporal regulation of hordeins and HvPDIL1-1. These results are discussed in relation to the putative role of HvPDIL1-1 in end-product quality in barley.

Expression of Genes for Si Uptake, Accumulation, and Correlation of Si with Other Elements in Ionome of Maize Kernel.

The mineral composition of cells, tissues, and organs is decisive for the functioning of the organisms, and is at the same time an indicator for understanding of physiological processes. We measured the composition of the ionome in the different tissues of maize kernels by element microanalysis, with special emphasis on silicon (Si). We therefore also measured the expression levels of the Si transporter genes ZmLsi1, ZmLsi2 and ZmLsi6, responsible for Si uptake and accumulation. Two weeks after pollination ZmLsi1 and ZmLsi6 genes were expressed, and expression continued until the final developmental stage of the kernels, while ZmLsi2 was not expressed. These results suggest that exclusively ZmLsi1 and ZmLsi6 are responsible for Si transport in various stages of kernel development. Expression level of ZmLsi genes was consistent with Si accumulation within kernel tissues. Silicon was mainly accumulated in pericarp and embryo proper and the lowest Si content was detected in soft endosperm and the scutellum. Correlation linkages between the distribution of Si and some other elements (macroelements Mg, P, S, N, P, and Ca and microelements Cl, Zn, and Fe) were found. The relation of Si with Mg was detected in all kernel tissues. The Si linkage with other elements was rather specific and found only in certain kernel tissues of maize. These relations may have effect on nutrient uptake and accumulation.

Ultrastructure of five Euglena species positioned in the subdivision Serpentes.

Within the genus Euglena, the subgroup "Serpentes" is characterised by species with long, slim cell bodies, which move without flagellum by snake-like locomotion in the detritus or in the mud, or swim freely in the water with a flagellum. Two major groups can be distinguished. The first is centred around the species Euglena satelles, with Euglena carterae, Euglena adhaerens and others, and is characterised by a straight-ended anterior part of the cell without a protruding flagellum. The second group is centred around the species Euglena deses, with its varieties, and Euglena ehrenbergii, and is characterised by a lateral canal opening at the anterior end with one flagellum protruding sideways. The representatives of the whole Serpentes group have various (15-30) large chloroplasts containing characteristic naked pyrenoids. The exception is Euglena ehrenbergii, which possesses innumerable small chloroplasts without pyrenoids. To better characterise this whole subgroup, to better taxonomically distinguish between the diverse species and to provide a basis for further molecular-genetic analysis of the phylogeny of and relationship between the Euglena species, we used transmission and scanning electron microscopy to investigate the five selected species. One important distinguishing feature among the species is the form of the pellicle. It can differ in thickness or cross-sectional shape (e.g. A-, M-or plateau-like shape) and can have various arrangements of microtubules and endoplasmic reticulum mucus vesicles. We show that the group is more heterogeneous than expected and that some species have very individual features that poorly fit into a common Serpentes group, particularly the above-mentioned Euglena ehrenbergii. Euglena carterae, formerly named Euglena deses var. carterae, with its typical straight-ended canal opening, does not fit into the Euglena deses varieties, as has already been confirmed by molecular genetic methods.