Biology subjects

Berendzen, K. W.

Publications and source records attributed to Berendzen, K. W..

4 recordsLinked to original sources

bioRxiv2023

A Tyrosine Phospho-switch within the Longin Domain of VAMP721 modulates SNARE functionality

The final step in secretion is membrane fusion facilitated by SNARE proteins that reside in opposite membranes. The formation of a trans-SNARE complex between one R and three Q coiled-coiled SNARE domains drives the final approach of the membranes providing the mechanical energy for fusion. Biological control of this mechanism is exerted by additional domains within some SNAREs. For example, the N-terminal Longin domain (LD) of R-SNAREs (also called Vesicle-associated membrane proteins, VAMPs) can fold back onto the SNARE domain blocking interaction with other cognate SNAREs. The LD may also determine the subcellular localization via interaction with other trafficking related proteins. Here, we provide cell-biological and genetic evidence that phosphorylation of the Tyrosine57 residue regulates the functionality of VAMP721. We found that an aspartate mutation mimics phosphorylation, leading to protein instability and subsequent degradation in lytic vacuoles. The mutant SNARE also fails to rescue the defects of vamp721vamp722 loss-of-function lines in spite of its wildtype-like localization within the secretory pathway and the ability to interact with cognate SNARE partners. Most importantly, it imposes a dominant negative phenotype interfering with root growth, normal secretion and cytokinesis in wildtype plants generating large aggregates that mainly contain secretory vesicles. Non-phosphorylatable VAMP721Y57F needs higher gene dosage to rescue double mutants in comparison to native VAMP721 underpinning that phosphorylation modulates SNARE function. We propose a model where a short-lived phosphorylation of Y57 serves as a regulatory step to control VAMP721 activity, favouring its open state and interaction with cognate partners to ultimately drive membrane fusion.

plant biology↗

bioRxiv2023

nf-root: a best-practice pipeline for deep learning-based analysis of apoplastic pH in microscopy images of developmental zones in plantroot tissue

Here we report nextflow-root (nf-root), a novel best-practice pipeline for deep learning-based analysis of fluorescence microscopy images of plant root tissue, aimed at studying hormonal mechanisms associated with cell elongation, given the vital role that plant hormones play in the development and growth of plants. This bioinformatics pipeline performs automatic identification of developmental zones in root tissue images, and analysis of apoplastic pH measurements of tissue zones, which is useful for modeling plant hormone signaling and cell physiological responses. Mathematical models of physiological responses of plant hormones, such as brassinolide, have been successfully established for certain root tissue types, by evaluating apoplastic pH via fluorescence imaging. However, the generation of data for this modeling is time-consuming, as it requires the manual segmentation of tissue zones and evaluation of large amounts of microscopy data. We introduce a high-throughput, highly reproducible Nextflow pipeline based on nf-core standards that automates tissue zone segmentation by implementing a deep-learning module, which deploys deterministically trained (i.e. bit-exact reproducible) convolutional neural network models, and augments the segmentation predictions with measures of prediction uncertainty and model interpretability, aiming to facilitate result interpretation and verification by experienced plant biologists. To train our segmentation prediction models, we created a publicly available dataset composed of confocal microscopy images of A. thaliana root tissue using the pH-sensitive fluorescence indicator, and manually annotated segmentation masks that identify relevant tissue zones. We applied this pipeline to analyze exemplary data, and observed a high statistical similarity between the manually generated results and the output of nf-root. Our results indicate that this approach achieves near human-level performance, and significantly reduces the time required to analyze large volumes of data, from several days to hours.

bioinformatics↗

bioRxiv2023

Cytokinins regulate spatially-specific ethylene production to control root growth in Arabidopsis

The two principal growth regulators cytokinins and ethylene are known to interact in the regulation of plant growth. However, information about underlying molecular mechanism and positional specificity of the cytokinin/ethylene crosstalk in root growth control is scarce. We have identified spatial specificity of cytokinin-regulated root elongation and root apical meristem (RAM) size, both of which we demonstrate to be ethylene biosynthesis-dependent. Upregulation of the cytokinin biosynthetic gene ISOPENTENYLTRANSFERASE (IPT) in proximal and peripheral tissues leads to both root and RAM shortening. In contrast, IPT activation in distal and inner tissues reduces RAM size while leaving the root length comparable to mock-treated controls. We show that cytokinins regulate two steps specific to ethylene biosynthesis, the production of ACC by ACC SYNTHASEs (ACSs), and its conversion to ethylene by ACC OXIDASEs (ACOs). We describe cytokinin- and ethylene-specific regulation controlling the activity of ACSs and ACOs that are spatially discrete along both proximo/distal and radial root axes. Using direct ethylene measurements, we identify ACO2, ACO3 and ACO4 as being responsible for ethylene biosynthesis and the ethylene-regulated root and RAM shortening in cytokinin-treated Arabidopsis. Finally, we describe the tight cooperation between cytokinin and ethylene signaling in cytokinin-induced, ethylene-regulated control of ACO4 due to the direct interaction between ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), a member of the multistep phosphorelay cascade and the C-terminal portion of ETHYLENE INSENSITIVE 2 (EIN2-C), a key regulator of canonical ethylene signaling.

plant biology↗

bioRxiv2022

Canalization of genome-wide transcriptional activity in Arabidopsis thaliana accessions by MET1-dependent CG methylation

BACKGROUNDEukaryotes employ epigenetic marks such as DNA methylation at cytosines both for gene regulation and genome defense. In Arabidopsis thaliana, a central role is played by methylation in the CG context, with profound effects on gene expression and transposable element (TE) silencing. Nevertheless, despite its conserved role, genome-wide CG methylation differs substantially between wild A. thaliana accessions. RESULTSWe hypothesized that global reduction of CG methylation would reduce epigenomic, transcriptomic and phenotypic diversity in A. thaliana accessions. To test our hypothesis, we knocked out MET1, which is required for CG methylation, in 18 early-flowering A. thaliana accessions. Homozygous met1 mutants in all accessions suffered from a range of common developmental defects such as dwarfism and delayed flowering, in addition to accession-specific abnormalities in rosette leaf architecture, silique morphology and fertility. Integrated analysis of genome-wide methylation, chromatin accessibility and transcriptomes confirmed that inactivation of MET1 greatly reduces CG methylation and alters chromatin accessibility at thousands of loci. While the effects on TE activation were similarly drastic in all accessions, the quantitative effects on non-TE genes varied greatly. The expression profiles of accessions became considerably more divergent from each other after genome-wide removal of CG methylation, although the expression of genes with diverse expression profiles across wild-type accessions tended to become more similar in mutants. CONCLUSIONSOur systematic analysis of MET1 requirement for genome function in different A. thaliana accessions revealed a dual role for CG methylation: for many genes, CG methylation appears to canalize expression levels, with methylation masking regulatory divergence. However, for a smaller subset of genes, CG methylation increases expression diversity beyond genetically encoded differences.

plant biology↗