Biology subjects

Spielmann, J.

Publications and source records attributed to Spielmann, J..

3 recordsLinked to original sources

bioRxiv2025

Broad application of plant protein tagging with ALFA tag for nanobody-based imaging and biochemical approaches

Epitope tags are broadly used for detecting, modifying or purifying proteins of interest, but their breadth of application is often rather poor. Recently, the rationally designed ALFA tag and its ALFA Nanobody complements the repertoire of epitope tags and emerged as a highly versatile epitope tag characterized in various animal models and that outperforms existing tags. Here we evaluated the ALFA tag/ALFA Nanobody technology in plants and showcase its application for in planta protein detection across multiple compartments or cellular structures, protein:protein interaction studies, protein purification, induced-proximity approaches, and super-resolution microcopy. Most importantly, we shed light on the edge of ALFA tagging technology for proteins difficult to tag for topological or functionals reasons. We provide the proof of concept of ALFA tag technology for the detection and functional analysis of the Arabidopsis IRT1 iron transporter. Overall, such versatile and validated toolbox of ALFA tag and ALFA Nanobody applications will serve as a resource of great interest for functional studies in plants.

plant biology↗

bioRxiv2024

Cis-regulatory mutations co-opting circadian clock regulation underlie naturally selected extreme trait in Arabidopsis halleri

HEAVY METAL ATPase 4 (HMA4) is required for the naturally selected traits of zinc/cadmium hyperaccumulation and hypertolerance of in Arabidopsis halleri. Cis-regulatory alterations and tandem triplication of AhHMA4 result in substantially elevated transcript levels compared to the closely related non-tolerant non-hyperaccumulator Arabidopsis thaliana. Here we identify cis-regulatory Metal Hyperaccumulation Elements (MHEs) necessary for AhHMA4 promoter activity, employing sequence comparisons and motif elicitation analyses combined with progressive deletions and site-directed mutagenesis of promoter-reporter constructs. We report that the promoters of all AhHMA4 gene copies share a distal MHE1 (consensus TGTAAC), and a proximal pair of MHE2s identical or highly similar to the Evening Element (AAAATATCT). Evening elements are known binding sites of Arabidopsis CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1), a phytochrome-regulated transcription factor in the core circadian clock. We show that the promoter of each AhHMA4 gene copy, but not of AtHMA4, mediates enhanced transcript levels of the reporter and their diel rhythmicity. These functional characteristics are CCA1-dependent and recapitulated by a synthetic reporter construct placing the MHE2 pair into the AtHMA4-promoter sequence context, according to the example of the AhHMA4-1 promoter. Consistent with our observations in transgenic reporter lines, AhHMA4 transcript levels follow a diel rhythm in wild-type A. halleri plants. Different from A. halleri, we identify complex repressive functionalities co-localizing with an upstream lncRNA and an intron in the 5 untranslated region of A. thaliana HMA4. In summary, our work exemplifies how cis-regulatory mutations contributed to the evolution of extreme physiological traits through the co-option of the circadian clock regulatory network.

plant biology↗

bioRxiv2022

Cadmium-induced endocytosis of the broad spectrum root metal transporter of Arabidopsis

Iron is an essential micronutrient for plant growth and development. Under low iron conditions, Arabidopsis plants take up soil iron using the root iron transporter IRT1. In addition to iron, IRT1 also transports others divalent metals including cadmium that consequently accumulates into plant tissues and enters the food chain. IRT1 expression was shown to be regulated at the transcriptional and post-translational levels by its essential metal substrates to maximize iron uptake while limiting the accumulation of zinc, manganese or cobalt. Here, we characterized the regulation of IRT1 by cadmium and uncovered a cadmium-mediated downregulation of IRT1 protein by endocytosis. A short term exposure to cadmium indeed decreased the celle surface levels of IRT1 through endocytosis and degradation. This is mediated through the direct binding of cadmium to histidine residues within the regulatory loop of IRT1. Moreover, we demonstrated that cadmium-induced IRT1 degradation uses ubiquitin-mediated endocytosis driven by the IDF1 E3 ligase. Altogether, this work sheds light on the mechanisms of cadmium-mediated downregulation of IRT1 and offers a unique opportunity to boost plant cadmium uptake in phytoremediation/phytoextraction strategies.

plant biology↗