Knopf, M. | Search bioRxiv

bioRxiv2025

A three amino acid sequence gates protein stability control of bHLH104 and iron uptake in Arabidopsis thaliana

Iron (Fe) homeostasis is regulated to prevent iron imbalance, with the help of redundant basic helix-loop-helix (bHLH) IVc transcription factors that can be controlled by Fe-binding E3 ligases such as BRUTUS (BTS) in Arabidopsis thaliana. However, knowledge gaps remain to fully explain the mechanistic basis of this redundant protein interaction module. The C-terminus of bHLH104 interacts with BTS. Structural predictions suggest involvement of the three terminal amino acids, proline-alanine-alanine (PAA). However, the importance of the PAA short sequence for post-translational regulation of bHLH104 and resulting plant phenotypes has not been experimentally tested. Here, we demonstrate that transgenic plants expressing a bHLH104 variant lacking PAA (b104^dPAA) constitutively upregulated Fe acquisition and Fe transport genes and consequently accumulated Fe in contrast to wild-type bHLH104 protein-expressing plants. The extent of Fe accumulation in independent b104^dPAA lines correlated positively with b104^dPAA protein abundance. In contrast to the wild-type bHLH104 form, there was no indication that b104^dPAA interacted with BTS or was ubiquitinated by it. Together, these findings corroborated that the PAA sequence is required for post-translational control of bHLH104 by BTS. Hence, targeted manipulation of bHLHIVc protein PAA sequence may represent a strategy for crop biofortification. Plain language summaryPlants tightly control iron levels. In Arabidopsis thaliana, transcription factor bHLH104 steers iron uptake. E3 ligases like BTS regulate bHLH104. We show that a three-amino acid terminal sequence of bHLH104 is needed for its targeting by the BTS pathway. bHLH104 lacking PAA leads to iron accumulation. Targeting this short sequence can be a strategy for crop biofortification.

plant biology↗

bioRxiv2025

An affordable and non-invasive validated machine-aided phenotyping pipeline identifies phenotypic variation of stress resilience in alkaline calcareous soil across the life cycle in Arabidopsis thaliana

Alkaline calcareous soils (ACS) are prevalent globally and challenge plant growth by limiting nutrient uptake, such as iron. The model plant Arabidopsis thaliana thrives in disturbed urban environments wherein ACS conditions frequently occur. Existing research largely focused on vegetatively grown A. thaliana, while there is a notable lack of studies examining phenotypic variations across the life cycle in ACS. A valuable tool for understanding plant stress resilience is machine-aided phenotyping as it is non-invasive, rapid and accurate. But it is often unavailable to individual plant labs. Here, we established and validated an affordable MicroScan with PlantEye-based machine-aided phenotyping approach, collected and correlated quantitative growth data across plant life cycles in response to ACS. We used A. thaliana wild type and the chlorotic coumarin-deficient mutant f6h1-1 to assess weekly morphological and leaf color data both manually and using a multispectral PlantEye device. Through correlation analysis, we selected machine parameters to differentiate size and leaf chlorosis phenotypes. The correlation analysis indicated a close connection between rosette size and multiple spectral parameters, highlighting the importance of the rosette size for plant growth. Most reliable phenotyping was at the beginning bolting stage. This methodology further is validated to detect novel leaf chlorosis phenotypes of known iron deficiency mutants across growth stages. This affordable machine-aided phenotyping procedure is suitable for high-throughput accurate screening of small-grown rosette plants, such as A. thaliana, and enables the discovery of novel genetic and phenotypic variation during the life cycle for understanding plant resilience in challenging soil environments. Short summary sentenceA PlantEye machine-aided non-invasive accurate and reliable phenotyping pipeline depicted the importance of the rosette size for phenotyping and detected leaf chlorosis phenotypes of A. thaliana mutants across the life-cycle on alkaline calcareous soil. Highlights and major findings- A MicroScan PlantEye machine-aided non-invasive phenotyping pipeline was established for assessing growth data of A. thaliana across the life cycle on alkaline calcareous soil and distinguishing leaf chlorosis phenotypes. - Rosette size was found an important trait that characterizes A. thaliana growth. - Machine phenotyping was most reliable at the beginning bolting stage. - New phenotypes were detected for Fe homeostasis mutants.

plant biology↗