Biology subjects

Knoblauch, M.

Publications and source records attributed to Knoblauch, M..

6 recordsLinked to original sources

bioRxiv2024

Anatomics MLT, an AI tool for large scale quantification of ultrastructural traits

The ever increasing breadth of biological knowledge has led to recent efforts to combine information from various fields into cell- or tissue atlases. Anatomical features are the structural basis for such efforts, but unfortunately large scale analysis of subcellular anatomical traits is currently a missing feature. Similarly, small phenotypic alterations of organelle- or cell-specific anatomical traits, such as an increase of the total volume or the number of mitochondria in response to certain stimuli, are currently hard to quantify. To provide tools to extract quantitative information from available 3D microscopic datasets generated with methods such as serial block face scanning electron microscopy we a) developed much improved fixation and embedding protocols for plants to drastically reduce processing artifacts and b) generated an easy-to-use AI tool for quantitative analysis and visualization of large-scale data sets. We make this tool available as open source.

cell biology↗

bioRxiv2024

Electric charge controls plasmodesma conductivity

While plant cells are enclosed by rigid cell walls that counteract intracellular hydrostatic pressure1, their plasma membrane, cytosol, and endoplasmic reticulum (ER) remain connected through plasmodesmata, nanoscopic cell wall pores2. Plasmodesmal cell-to-cell transport occurs in the cytosolic sleeve between the plasma membrane and the ER membrane3-5, and is generally thought to be limited by the size of the moving particle alone6. Given that biological membranes carry negative electric surface charges7-9, this steric notion conflicts with physical theory of ion diffusion in nanometer-sized pores with charged walls10. Quantifying the movements of differently sized and charged fluorescent dyes in Tradescantia stamen hairs, we found that anionic fluorophores of up to 1 kDa traversed plasmodesmata whereas much smaller cationic ones did not. While this agrees with theoretical expectations of different size exclusion limits for cations and anions, it questions current dogma concerning plasmodesma function and also structure, as it implies positively rather than negatively charged surfaces within plasmodesmal pores. Our findings call for re-evaluations of current models of symplasmic transport, especially of charged molecules like the phytohormone auxin (indole-acetic acid) and certain amino acids.

cell biology↗

bioRxiv2024

Symplasmic phloem loading and sub-cellular transport in storage roots are key factors for carbon allocation in cassava

Cassava is a deciduous woody perennial shrub that stores large amounts of carbon and water in its storage roots. Previous studies have shown that assimilate unloading into storage roots happens symplasmically once secondary anatomy is established. However, mechanisms controlling phloem loading and overall carbon partitioning to different cassava tissues remain unclear. Here we used a combination of histological, transcriptional, and biochemical analyses on different cassava tissues and timepoints to better understand source-sink carbon allocation. We find that cassava likely utilizes a predominantly passive symplasmic phloem loading strategy, indicated by the lack of expression of genes coding for key players of sucrose transport, the existence of branched plasmodesmata in the companion cell/bundle sheath interface of minor leaf veins, and very high leaf sucrose concentrations. Furthermore, we show that tissue-specific changes in anatomy and NSC contents are associated with tissue-specific modification in gene expression for sucrose cleavage/synthesis, as well as subcellular compartmentalization of sugars. Overall, our data suggest that carbon allocation during storage root filling is mostly facilitated symplasmically, and is likely mostly regulated by local tissue demand and subcellular compartmentalization.

plant biology↗

bioRxiv2021

Maize Brittle Stalk2-Like3, encoding a COBRA protein, functions in cell wall formation and carbohydrate partitioning

Carbohydrate partitioning from leaves to sink tissues is essential for plant growth and development. The maize (Zea mays) recessive carbohydrate partitioning defective28 (cpd28) and cpd47 mutants exhibit leaf chlorosis and accumulation of starch and soluble sugars. Transport studies with 14C-sucrose (Suc) found drastically decreased export from mature leaves in cpd28 and cpd47 mutants relative to wild-type siblings. Consistent with decreased Suc export, cpd28 mutants exhibited decreased phloem pressure in mature leaves, and altered phloem cell wall ultrastructure in immature and mature leaves. We identified the causative mutations in the Brittle Stalk2-Like3 (BK2L3) gene, a member of the COBRA family, which is involved in cell wall development across angiosperms. None of the previously characterized COBRA genes are reported to affect carbohydrate export. Consistent with other characterized COBRA members, the BK2L3 protein localized to the plasma membrane, and the mutants condition a dwarf phenotype in dark-grown shoots and primary roots, as well as the loss of anisotropic cell elongation in the root elongation zone. Likewise, both mutants exhibit a significant cellulose deficiency in mature leaves. Therefore, BK2L3 functions in tissue growth and cell wall development, and this work elucidates a unique connection between cellulose deposition in the phloem and whole-plant carbohydrate partitioning. Funding InformationThe research was supported by US National Science Foundation Plant Genome Research Program grants (IOS-1025976 and IOS-1444448) to DMB.

genetics↗

bioRxiv2021

Diversity of funnel plasmodesmata in angiosperms: the impact of geometry on plasmodesmal resistance

In most plant tissues, threads of cytoplasm, or plasmodesmata, connect the protoplasts via pores in the cell walls. This enables symplasmic transport, for instance in phloem loading, transport, and unloading. Importantly, the geometry of the wall pore limits the size of the particles that may be transported, and also (co-)defines plasmodesmal resistance to diffusion and convective flow. However, quantitative information on transport through plasmodesmata in non-cylindrical cell wall pores is scarce. We have found conical, funnel-shaped cell wall pores in the phloem-unloading zone in growing root tips of five eudicot and two monocot species, specifically between protophloem sieve elements and phloem pole pericycle cells. 3D reconstructions by electron tomography suggested that funnel plasmodesmata possess a desmotubule but lack tethers to fix it in a central position. Model calculations showed that both diffusive and hydraulic resistance decrease drastically in conical cell wall pores compared to cylindrical channels, even at very small opening angles. Notably, the effect on hydraulic resistance was relatively larger. We conclude that funnel plasmodesmata generally are present in specific cell-cell interfaces in angiosperm roots, where they appear to facilitate symplasmic phloem unloading. Interestingly, cytosolic sleeves of most plasmodesmata reported in the literature do not resemble straight annuli but possess variously shaped widenings. Our evaluations suggest that widenings too small for identification on electron micrographs may drastically reduce the hydraulic and diffusional resistance of these pores. Consequently, theoretical models assuming cylindrical symmetries will underestimate plasmodesmal conductivities.

plant biology↗

bioRxiv2018

Protein Structural Biology Using Cell-Free Platform from Wheat Germ

One of the biggest bottlenecks for structural analysis of proteins remains the creation of high yield and high purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multistage eukaryotic wheat-germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also developed a robust purification procedure, which can produce highly-pure (>98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid generation of samples for structural characterization that may not be amenable to other recombinant approaches.

molecular biology↗