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Plant breeders leverage mutagenesis using chemical, biological, and physical mutagens to create novel trait variations. Many widely used sorghum genotypes have a narrow genetic base, which hinders improvements using classical breeding. Enhancing the diversity of the sorghum genome thus remains a key priority for sorghum breeders. To accelerate the genetic enhancement of sorghum, an extensive library comprised of seeds from 150,000 individual mutant plants of the Sorghum bicolor inbred line BTx623 was established using ethyl methanesulphonate (EMS) as a mutagen. The sorghum mutant library was bulked into 1498 pools (~100 seed heads per pool). In each pool, DNA was extracted from a subset of the seed and screened using the FIND‐IT technology based on droplet digital PCR. All 43 nucleotide substitutions that were screened using FIND‐IT were identified, demonstrating the potential to identify any EMS‐derived mutation in an elite line of sorghum within days. This diverse library represents the largest collection of sorghum mutants ever conceived, estimated to cover 240% of all possible EMS‐induced mutation points within the Sorghum genome. Using FIND‐IT, the speed at which a specific desired EMS‐derived mutation can be identified is a major upgrade to conventional reverse genetic techniques. Additionally, the ease at which valuable variants can be integrated into elite commercial lines is a far simpler and less expensive process compared to genome editing. Genomic variations in the library will have direct utility as a breeding resource for commercial sorghum applications, allowing enhanced adaptation to climate change and enhanced yield potential in marginal environments. [ABSTRACT FROM AUTHOR]

The epidermis on leaves protects plants from pathogen invasion and provides a waterproof barrier. It consists of a layer of cells that is surrounded by thick cell walls, which are partially impregnated by highly hydrophobic cuticular components. We show that the Arabidopsis T-DNA insertion mutants of REDUCED WALL ACETYLATION 2 (rwa2), previously identified as having reduced O-acetylation of both pectins and hemicelluloses, exhibit pleiotrophic phenotype on the leaf surface. The cuticle layer appeared diffused and was significantly thicker and underneath cell wall layer was interspersed with electron-dense deposits. A large number of trichomes were collapsed and surface permeability of the leaves was enhanced in rwa2 as compared to the wild type. A massive reprogramming of the transcriptome was observed in rwa2 as compared to the wild type, including a coordinated up-regulation of genes involved in responses to abiotic stress, particularly detoxification of reactive oxygen species and defense against microbial pathogens (e.g., lipid transfer proteins, peroxidases). In accordance, peroxidase activities were found to be elevated in rwa2 as compared to the wild type. These results indicate that cell wall acetylation is essential for maintaining the structural integrity of leaf epidermis, and that reduction of cell wall acetylation leads to global stress responses in Arabidopsis.

Manganese (Mn) toxicity is a major problem for plant growth, often caused by acidic soils leading to an excessive plant availability of Mn2+. However, Mn deficiency is also a major nutritional disorder, commonly observed on slightly alkaline soils, which are favoring oxidation of soluble Mn2+ to the plant unavailable form MnO2. The work reported here was initiated with the aim of identifying and characterizing barley Mn2+ transport proteins. Using yeast as a model system we have identified and characterized barley genes encoding Mn2+ transport proteins. The plasma membrane localized HvIRT1 protein is a broad range metal ion transporter. The HvIRT1 transcript expression was induced under Mn and Fe deficiency and the expression level was correlated with the Mn2+ uptake capacity, suggesting an important role of HvIRT1 in Mn2+ acquisition. Two additional genes encoding Mn2+ transport proteins were identified in a Mn2+ tolerance yeast assay; one belonging to the CDF family designated HvMTP8.1 and one homologous gene to the yeast CCC1, named HvVIT1. It is proposed that HvMTP8.1 has a role in Mn2+ efflux mechanisms into the apoplast whereas HvVIT1 are involved in Mn2+ sequestration into internal cellular compartments. The results presented provide the basis for an improved understanding of the mechanisms involved in Mn2+ acquisition and homeostasis in crop plants. This information will facilitate engineering of genotypes able to grow efficiently on marginal soils.

Most living organisms express a number of small cysteine-rich metalloproteins with strong binding capacity of transition metals, which are known as metallothioneins (MTs). Plant MTs are differentially expressed in various plant organs, as well as developmentally regulated and inducible in response to a wide variety of environmental stimuli and stressors. However, there is still limited knowledge regarding their physiological functions in plants. Here we present some fundamental steps towards a functional characterisation of a barley (Hordeum vulgare) type 2 metallothionein promoter in transgenic Arabidopsis thaliana.

Abstract Background Quantitative multi-elemental analysis by inductively coupled plasma (ICP) spectrometry depends on a complete digestion of solid samples. However, fast and thorough sample digestion is a challenging analytical task which constitutes a bottleneck in modern multi-elemental analysis. Additional obstacles may be that sample quantities are limited and elemental concentrations low. In such cases, digestion in small volumes with minimum dilution and contamination is required in order to obtain high accuracy data. Results We have developed a micro-scaled microwave digestion procedure and optimized it for accurate elemental profiling of plant materials (1-20 mg dry weight). A commercially available 64-position rotor with 5 ml disposable glass vials, originally designed for microwave-based parallel organic synthesis, was used as a platform for the digestion. The novel micro-scaled method was successfully validated by the use of various certified reference materials (CRM) with matrices rich in starch, lipid or protein. When the micro-scaled digestion procedure was applied on single rice grains or small batches of Arabidopsis seeds (1 mg, corresponding to approximately 50 seeds), the obtained elemental profiles closely matched those obtained by conventional analysis using digestion in large volume vessels. Accumulated elemental contents derived from separate analyses of rice grain fractions (aleurone, embryo and endosperm) closely matched the total content obtained by analysis of the whole rice grain. Conclusion A high-throughput micro-scaled method has been developed which enables digestion of small quantities of plant samples for subsequent elemental profiling by ICP-spectrometry. The method constitutes a valuable tool for screening of mutants and transformants. In addition, the method facilitates studies of the distribution of essential trace elements between and within plant organs which is relevant for, e.g., breeding programmes aiming at improvement of the micronutrient density in edible plant parts. Compared to existing vial-in-vial systems, the new method developed here represents a significant methodological advancement in terms of higher capacity, reduced labour consumption, lower material costs, less contamination and, as a consequence, improved analytical accuracy following micro-scaled digestion of plant samples.

Summary: Transporters involved in manganese (Mn) uptake and intracellular Mn homeostasis in Arabidopsis and rice are well characterized, while much less is known for barley, which is particularly prone to Mn deficiency. In this study we have investigated the role of the iron‐regulated transporter 1 (IRT1) for Mn uptake and translocation in barley plants. We employed an RNAi approach to reduce HvIRT1 expression to 5% of the wild‐type level. This enabled characterization of the functional role of HvIRT1 by use of advanced imaging and phenotyping techniques applied to plants growing in hydroponics or soils with different Mn availability. Our results highlight the importance of HvIRT1 for the transport of Mn across the root endodermis into the stele. In the hvirt1‐RNAi lines, a chlorotic phenotype with reduced shoot Mn concentration and impaired photosynthetic functionality was observed, especially under conditions with low Mn availability. We also document that HvIRT1 controlled the Mn distribution within the barley grain. Surprisingly, unlike other IRT1 orthologues, HvIRT1 played no significant role in iron uptake. We conclude that the barley IRT1 orthologue has a novel function with respect to ensuring sufficient shoot Mn concentrations. The preference of IRT1 for Mn instead of Fe is discussed in an evolutionary context. [ABSTRACT FROM AUTHOR]