A bench-top Dark-Root device built with LEGO® bricks enables a non-invasive plant root development analysis in soil conditions mirroring nature.

Roots are the hidden parts of plants, anchoring their above-ground counterparts in the soil. They are responsible for water and nutrient uptake and for interacting with biotic and abiotic factors in the soil. The root system architecture (RSA) and its plasticity are crucial for resource acquisition and consequently correlate with plant performance while being highly dependent on the surrounding environment, such as soil properties and therefore environmental conditions. Thus, especially for crop plants and regarding agricultural challenges, it is essential to perform molecular and phenotypic analyses of the root system under conditions as near as possible to nature (#asnearaspossibletonature). To prevent root illumination during experimental procedures, which would heavily affect root development, Dark-Root (D-Root) devices (DRDs) have been developed. In this article, we describe the construction and different applications of a sustainable, affordable, flexible, and easy to assemble open-hardware bench-top LEGO® DRD, the DRD-BIBLOX (Brick Black Box). The DRD-BIBLOX consists of one or more 3D-printed rhizoboxes, which can be filled with soil while still providing root visibility. The rhizoboxes sit in a scaffold of secondhand LEGO® bricks, which allows root development in the dark and non-invasive root tracking with an infrared (IR) camera and an IR light-emitting diode (LED) cluster. Proteomic analyses confirmed significant effects of root illumination on barley root and shoot proteomes. Additionally, we confirmed the significant effect of root illumination on barley root and shoot phenotypes. Our data therefore reinforces the importance of the application of field conditions in the lab and the value of our novel device, the DRD-BIBLOX. We further provide a DRD-BIBLOX application spectrum, spanning from investigating a variety of plant species and soil conditions and simulating different environmental conditions and stresses, to proteomic and phenotypic analyses, including early root tracking in the dark.

Identification of apple cultivars hypoallergenic for birch pollen-allergic individuals by a multidisciplinary in vitro and in vivo approach.

BACKGROUND: Birch pollen-related apple allergy is the most frequent IgE-mediated food allergy in Central-Northern Europe with Mal d 1 as major allergen. Its concentration in apples varies with the cultivar and storage time. Year-round appealing, hypoallergenic cultivars still are needed to satisfy the nutritional needs of affected individuals. We characterized three promising cultivars by multidisciplinary in vitro assays including long-term storage and by clinical challenges of allergic individuals before and after the birch pollen season. METHODS: Proteins were extracted from fruits of 'Santana', 'Golden Delicious' (GD), and three genuine cultivars in November 2018 and April 2019. Mal d 1-levels were analysed by mass spectrometry, SDS-PAGE, immunoblotting, competitive ELISA, and basophil activation tests. Twenty-eight allergic individuals underwent single-blinded open food challenges and skin testing with the cultivars and birch pollen in November 2018 and May 2019. Allergen-specific IgE-levels were determined. RESULTS: After storage all cultivars except 'Santana' were of appealing appearance and taste. Their Mal d 1 content had increased, also reflected by significantly amplified basophil activation and stronger reactions in clinical challenges. Besides, individuals showed boosted reactivity after pollen exposure indicated by enhanced allergen-specific IgE-levels and skin reactions to birch pollen. Still, all cultivars remained significantly less allergenic than GD and comparable to Santana in November 2018 in all assessments except for skin testing. CONCLUSIONS: Combined expertise in pomology and allergology identified promising new cultivars for allergic consumers. The evaluation of hypoallergenic apples should incorporate long-term storage and birch pollen exposure. Basophil activation tests may be suitable in the selection of promising cultivars for oral challenges.

Tissue-Specific Proteome and Subcellular Microscopic Analyses Reveal the Effect of High Salt Concentration on Actin Cytoskeleton and Vacuolization in Aleurone Cells during Early Germination of Barley.

Cereal grain germination provides the basis for crop production and requires a tissue-specific interplay between the embryo and endosperm during heterotrophic germination involving signalling, protein secretion, and nutrient uptake until autotrophic growth is possible. High salt concentrations in soil are one of the most severe constraints limiting the germination of crop plants, affecting the metabolism and redox status within the tissues of germinating seed. However, little is known about the effect of salt on seed storage protein mobilization, the endomembrane system, and protein trafficking within and between these tissues. Here, we used mass spectrometry analyses to investigate the protein dynamics of the embryo and endosperm of barley (Hordeum vulgare, L.) at five different early points during germination (0, 12, 24, 48, and 72 h after imbibition) in germinated grains subjected to salt stress. The expression of proteins in the embryo as well as in the endosperm was temporally regulated. Seed storage proteins (SSPs), peptidases, and starch-digesting enzymes were affected by salt. Additionally, microscopic analyses revealed an altered assembly of actin bundles and morphology of protein storage vacuoles (PSVs) in the aleurone layer. Our results suggest that besides the salt-induced protein expression, intracellular trafficking and actin cytoskeleton assembly are responsible for germination delay under salt stress conditions.

Protein sorting into protein bodies during barley endosperm development is putatively regulated by cytoskeleton members, MVBs and the HvSNF7s.

Cereal endosperm is a short-lived tissue adapted for nutrient storage, containing specialized organelles, such as protein bodies (PBs) and protein storage vacuoles (PSVs), for the accumulation of storage proteins. During development, protein trafficking and storage require an extensive reorganization of the endomembrane system. Consequently, endomembrane-modifying proteins will influence the final grain quality and yield. However, little is known about the molecular mechanism underlying endomembrane system remodeling during barley grain development. By using label-free quantitative proteomics profiling, we quantified 1,822 proteins across developing barley grains. Based on proteome annotation and a homology search, 94 proteins associated with the endomembrane system were identified that exhibited significant changes in abundance during grain development. Clustering analysis allowed characterization of three different development phases; notably, integration of proteomics data with in situ subcellular microscopic analyses showed a high abundance of cytoskeleton proteins associated with acidified PBs at the early development stages. Moreover, endosomal sorting complex required for transport (ESCRT)-related proteins and their transcripts are most abundant at early and mid-development. Specifically, multivesicular bodies (MVBs), and the ESCRT-III HvSNF7 proteins are associated with PBs during barley endosperm development. Together our data identified promising targets to be genetically engineered to modulate seed storage protein accumulation that have a growing role in health and nutritional issues.