73 research outputs found
Asymmetric wall ingrowth deposition in Arabidopsis phloem parenchyma transfer cells is tightly associated with sieve elements
In Arabidopsis, polarized deposition of wall ingrowths in phloem parenchyma (PP) transfer cells (TCs) occurs adjacent to cells of the sieve element/companion cell (SE/CC) complex. However, the spatial relationships between these different cell types in minor veins, where phloem loading occurs, are poorly understood. PP TC development and wall ingrowth localization were compared with those of other phloem cells in leaves of Col-0 and the transgenic lines AtSUC2::AtSTP9-GFP (green fluorescent protein) and AtSWEET11::AtSWEET11-GFP that identify CCs and PP cells, respectively. The development of PP TCs in minor veins, indicated by deposition of wall ingrowths, proceeded basipetally in leaves. However, not all PP cells develop wall ingrowths, and higher levels of deposition occur in abaxial- compared with adaxial-positioned PP TCs. Furthermore, the deposition of wall ingrowths was exclusively initiated on and preferentially covered the PP TC/SE interface, rather than the PP TC/CC interface, and only occurred in PP cells that were adjacent to SEs. Collectively, these results demonstrate a tight association between SEs and wall ingrowth deposition in PP TCs and suggest the existence of two subtypes of PP cells in leaf minor veins. Compared with PP cells, PP TCs showed more abundant accumulation of AtSWEET11–GFP, indicating functional differences in phloem loading between PP and PP TCs
Death receptor-based enrichment of Cas9-expressing cells
Background: The CRISPR/Cas9 genome editing system has greatly facilitated and expanded our capacity to engineer mammalian genomes, including targeted gene knock-outs. However, the phenotyping of the knock-out effect requires a high DNA editing efficiency. Results: Here, we report a user-friendly strategy based on the extrinsic apoptosis pathway that allows enrichment of a polyclonal gene-edited cell population, by selecting Cas9-transfected cells that co-express dominant-negative mutants of death receptors. The extrinsic apoptosis pathway can be triggered in many mammalian cell types, and ligands are easy to produce, do not require purification and kill much faster than the state-of-the-art selection drug puromycin. Stringent assessment of our advanced selection strategy via Sanger sequencing, T7 endonuclease I (T7E1) assay and direct phenotyping confirmed a strong and rapid enrichment of Cas9-expressing cell populations, in some cases reaching up to 100Â % within one hour. Notably, the efficiency of target DNA cleavage in these enriched cells reached high levels that exceeded the reliable range of the T7E1 assay, a conclusion that can be generalized for editing efficiencies above 30Â %. Moreover, our data emphasize that the insertion and deletion pattern induced by a specific gRNA is reproducible across different cell lines. Conclusions: The workflow and the findings reported here should streamline a wide array of future low- or high-throughput gene knock-out screens, and should largely improve data interpretation from CRISPR experiments
Passive phloem loading and long-distance transport in a synthetic tree-on-a-chip
Vascular plants rely on differences of osmotic pressure to export sugars from
regions of synthesis (mature leaves) to sugar sinks (roots, fruits). In this
process, known as M\"unch pressure flow, the loading of sugars from
photosynthetic cells to the export conduit (the phloem) is crucial, as it sets
the pressure head necessary to power long-distance transport. Whereas most
herbaceous plants use active mechanisms to increase phloem concentration above
that of the photosynthetic cells, in most tree species, for which transport
distances are largest, loading seems to occur via passive symplastic diffusion
from the mesophyll to the phloem. Here, we use a synthetic microfluidic model
of a passive loader to explore the nonlinear dynamics that arise during export
and determine the ability of passive loading to drive long-distance transport.
We first demonstrate that in our device, phloem concentration is set by the
balance between the resistances to diffusive loading from the source and
convective export through the phloem. Convection-limited export corresponds to
classical models of M\"unch transport, where phloem concentration is close to
that of the source; in contrast, diffusion-limited export leads to small phloem
concentrations and weak scaling of flow rates with the hydraulic resistance. We
then show that the effective regime of convection-limited export is predominant
in plants with large transport resistances and low xylem pressures. Moreover,
hydrostatic pressures developed in our synthetic passive loader can reach
botanically relevant values as high as 10 bars. We conclude that passive
loading is sufficient to drive long-distance transport in large plants, and
that trees are well suited to take full advantage of passive phloem loading
strategies
Symplasmic transport and phloem loading in gymnosperm leaves
Despite more than 130Â years of research, phloem loading is far from being understood in gymnosperms. In part this is due to the special architecture of their leaves. They differ from angiosperm leaves among others by having a transfusion tissue between bundle sheath and the axial vascular elements. This article reviews the somewhat inaccessible and/or neglected literature and identifies the key points for pre-phloem transport and loading of photoassimilates. The pre-phloem pathway of assimilates is structurally characterized by a high number of plasmodesmata between all cell types starting in the mesophyll and continuing via bundle sheath, transfusion parenchyma, Strasburger cells up to the sieve elements. Occurrence of median cavities and branching indicates that primary plasmodesmata get secondarily modified and multiplied during expansion growth. Only functional tests can elucidate whether this symplasmic pathway is indeed continuous for assimilates, and if phloem loading in gymnosperms is comparable with the symplasmic loading mode in many angiosperm trees. In contrast to angiosperms, the bundle sheath has properties of an endodermis and is equipped with Casparian strips or other wall modifications that form a domain border for any apoplasmic transport. It constitutes a key point of control for nutrient transport, where the opposing flow of mineral nutrients and photoassimilates has to be accommodated in each single cell, bringing to mind the principle of a revolving door. The review lists a number of experiments needed to elucidate the mode of phloem loading in gymnosperms
3D micro-macro fluid-structure model of pressure relief valve leak tightness
Controlling and assessing the leak tightness of a Pressure Relief Valve (PRV) has been a challenge since the original design of the product. With more stringent demands from the nu- clear power industry for leakproof PRV’s, closer to the set point, there has been a drive by both industry and academia for a better design method for many known metal-to-metal contacting seal/surface problems. This paper outlines a numerical modelling strategy drawn from industry experience and metrology measurements and investigates the effects of lapping and surface finish on leakage rate. Key influencing parameters of surface form, waviness and roughness are incorporated in the analysis. The numerical approach requires efficient coupling of a non-linear structural Finite Element Analysis (FEA) with a Computational Fluid Dynamic (CFD) solver. This allows the examination of the relationship between deformation of the contacting surfaces, based on the applied spring force, and the resulting micro-flow of gas through any available gaps and the overall leakage to be found. The API527 Seat Tightness methodology is followed to allow leakage rates to be measured and the computational model to be preliminarily validated. Using this model, engineers can adjust and optimise the design of pressure relief valves to find the minimal leakage condition for a given configuration. In addition, the numerical approach can potentially be applied to other metal-to-metal contacting surface components, such as flanges with metal gaskets, and help eliminate leakage
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