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 13 C-CO 2 -labeled atmosphere, while 15 N-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 13 C-CO 2 -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 13 C 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.

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.

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).

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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.

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.

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.

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