Bioinformatics-based assessment of the relevance of candidate genes for mutation discovery

The bioinformatics resources provide a wide range of tools that can be applied in different areas of mutation screening. The enormous and constantly increasing amount of genomic data obtained in plant-oriented molecular studies requires the development of efficient techniques for its processing. There is a wide range of bioinformatics tools which can aid in the course of mutation discovery. The following chapter focuses mainly on the application of different tools and resources to facilitate a Targeting-Induced Local Lesions in Genomes (TILLING) analysis. TILLING is a technique of reverse genetics that applies a traditional mutagenesis to create DNA libraries of mutagenised individuals that are then subjected to high-throughput screening for the identification of mutations. The bioinformatics tools have shown to be useful in supporting the process of candidate gene selection for mutation screening. The availability of bioinformatics software and experimental data repositories provides a powerful tool which enables a process of multi-database mining. The existing raw experimental data (genomics-related information, expression data, annotated ontologies) can be interpreted in terms of a new biological context. This may help in selecting the proper candidate gene for mutation discovery that is controlling the target phenotype. The mutation screening using a TILLING strategy requires a former knowledge of the full genomic sequence of the gene which is of interest. Depending on whether a fully sequenced genome of a particular species is available, different bioinformatics tools can facilitate this process. Specific tools can be also useful for the identification of possible gene paralogs which may mask the effect of mutated gene. Bioinformatics resources can also support the selection of gene fragments most prone to acquire a deleterious nucleotide change. Finally, there are available tools enabling a proper design of oligonucleotide primers for the amplification of a gene fragment for the purpose of mutation screening

New allele of HvBRI1 gene encoding brassinosteroid receptor in barley

The aim of these studies was to characterize nucleotide substitutions leading to the phenotype of brassinosteroid-insensitive, semi-dwarf barley mutant 093AR. Two substitutions in the sequence of barley HvBRI1 gene, encoding leucine-rich repeats receptor kinase (LRRRK), which participates in brassinosteroid (BR) signalling, were identified in this chemically-induced barley mutant of the cv. Aramir. The LRR-RK is a transmembrane protein phosphorylating downstream components. The identified substitutions CC>AA at positions 1760 and 1761 in the HvBRI1 gene of this mutant led to a missense mutation, causing the Thr-573 to Lys-573 replacement in the protein sequence. The threonine residue is situated in the distal part of a 70-amino acids island responsible for binding of BR molecules. As this residue is conserved among BRI1 protein homologs in Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa and Hordeum vulgare, it was postulated that this residue is crucial for the protein function. The genetic analyses indicated that the mutant 093AR was allelic to the spontaneous, semi-dwarf mutant uzu which carries A>G substitution at position 2612 of the HvBRI1 gene (GenBank acc. no. AB088206). A comparison of the genomic sequence of HvBRI1 in the mutants uzu, 093AR and in the cv. ‘Aramir’ confirmed the presence of the single-nucleotide A>G substitution at position 2612 in the sequence encoding kinase domain of HvBRI1 polypeptide in uzu, but not in 093AR mutant, indicating that a new allele of the HvBRI1 gene was identified

The barley EST DNA Replication and Repair Database (bEST-DRRD) as a tool for the identification of the genes involved in DNA replication and repair

Background: The high level of conservation of genes that regulate DNA replication and repair indicates that they may serve as a source of information on the origin and evolution of the species and makes them a reliable system for the identification of cross-species homologs. Studies that had been conducted to date shed light on the processes of DNA replication and repair in bacteria, yeast and mammals. However, there is still much to be learned about the process of DNA damage repair in plants.Description: These studies, which were conducted mainly using bioinformatics tools, enabled the list of genes that participate in various pathways of DNA repair in Arabidopsis thaliana (L.) Heynh to be outlined; however, information regarding these mechanisms in crop plants is still very limited. A similar, functional approach is particularly difficult for a species whose complete genomic sequences are still unavailable. One of the solutions is to apply ESTs (Expressed Sequence Tags) as the basis for gene identification. For the construction of the barley EST DNA Replication and Repair Database (bEST-DRRD), presented here, the Arabidopsis nucleotide and protein sequences involved in DNA replication and repair were used to browse for and retrieve the deposited sequences, derived from four barley (Hordeum vulgare L.) sequence databases, including the " Barley Genome version 0.05" database (encompassing ca. 90% of barley coding sequences) and from two databases covering the complete genomes of two monocot models: Oryza sativa L. and Brachypodium distachyon L. in order to identify homologous genes. Sequences of the categorised Arabidopsis queries are used for browsing the repositories, which are located on the ViroBLAST platform. The bEST-DRRD is currently used in our project during the identification and validation of the barley genes involved in DNA repair.Conclusions: The presented database provides information about the Arabidopsis genes involved in DNA replication and repair, their expression patterns and models of protein interactions. It was designed and established to provide an open-access tool for the identification of monocot homologs of known Arabidopsis genes that are responsible for DNA-related processes. The barley genes identified in the project are currently being analysed to validate their function

Identification of barley DWARF gene involved in brassinosteroid synthesis

Brassinosteroids (BR) are plant steroid hormones regulating various aspects of morphogenesis, such as seed development and germination, cell division and elongation, differentiation of tracheary elements, development during growth in darkness (skotomorphogenesis), photosynthesis and response to environmental stress. Brassinosteroid synthesis has been studied to a great extent in the dicot species, Arabidopsis thaliana, resulting in the identification of genes participating in this process. Much less is known about BR synthesis in crops, including the monocots. The purpose of this study was to identify and characterize barley coding sequence HvDWARF involved in brassinosteroid synthesis. This sequence, encoding brassinosteroid-6-oxidase, was identified on the basis of barley ESTs. This sequence was screened for nucleotide substitutions in semi-dwarf, chemically-induced barley mutants exhibiting changes in etiolation. The responsiveness of these genotypes to exogenous brassinosteroids was determined with the use of leaf-blade segment unrolling tests. The semi-dwarf phenotype of the BR-deficient mutants was rescued by the application of 10-5 M 24-epibrassinolide. Two missense mutations were identified within the HvDWARF sequence in BR-deficient mutants 522DK and 527DK from variety ‘Delisa’. These substitutions cause changes of amino acid residues located within the conserved fragments of the encoded polypeptide. Thetranscription profile of HvDWARF and HvBAK1/SERK3, involved in BR signaling, was determined during the early stages of seedling development in BR-deficient and BRinsensitive mutants using real-time quantitative PCR. This analysis indicated that HvDWARF displays a uniformly low level of this process, whereas the transcription level of HvBAK1 proved to be spatially and temporally regulated

An automated, cost-effective and scalable, flood-and-drain based root phenotyping system for cereals

Background: Genetic studies on the molecular mechanisms of the regulation of root growth require the characterisation of a specific root phenotype to be linked with a certain genotype. Such studies using classical labour-intensive methods are severely hindered due to the technical limitations that are associated with the impeded observation of the root system of a plant during its growth. The aim of the research presented here was to develop a reliable, cost-effective method for the analysis of a plant root phenotype that would enable the precise characterisation of the root system architecture of cereals. Results: The presented method describes a complete system for automatic supplementation and continuous sensing of culture solution supplied to plants that are grown in transparent tubes containing a solid substrate. The presented system comprises the comprehensive pipeline consisting of a modular-based and remotely-controlled plant growth system and customized imaging setup for root and shoot phenotyping. The system enables an easy extension of the experimental capacity in order to form a combined platform that is comprised of parallel modules, each holding up to 48 plants. The conducted experiments focused on the selection of the most suitable conditions for phenotyping studies in barley: an optimal size of the glass beads, diameters of the acrylic tubes, composition of a medium, and a rate of the medium flow. Conclusions: The developed system enables an efficient, accurate and highly repeatable analysis of the morphological features of the root system of cereals. Because a simple and fully-automated control system is used, the experimental conditions can easily be normalised for different species of cereals. The scalability of the module-based system allows its capacity to be adjusted in order to meet the requirements of a particular experiment

Root phenotyping pipeline for cereal plants

The proposed system for the phenotypic analysis of root traits that is presented here enables the precise description of the root growth kinetics of cereal plants. The designed pipeline is composed of a drip irrigation system to supplement plants with a medium, a high-resolution root system scanning facility and a method for comprehensive image analysis. The system enables low-effort, accurate and highly repeatable analysis of features of the root system of cereal seedlings and young plants until the early tillering stage. This system employs an automatic drip irrigation line, which is controlled remotely by a programmable logic controller (PLC). The PLC adapter used facilitates the automated control of all system modules, thus allowing the rate of the medium flow to be adjusted for the supplementation of plants. The system employs measuring sensors for the continuous monitoring of the parameters of the culture medium. This continuous sensing of medium parameters can be applicable for mineral nutrition studies and abiotic stress response testing. The installed drip lines are injected into transparent acrylic tubes (500 mm high, 32/30 mm in outer and inner diameter, with a circular opening in the bottom of 3 mm in diameter) that are filled with glass beads. The acrylic tubes are placed in opaque cover tubes that permit the non-destructive observation of the growth of the root system. Enhanced imaging quality contributes to an increase in the precision of the results that are obtained in the course of the analysis of root parameters using specialised root scanners coupled with the WinRHIZO system. This novel phenotyping pipeline permits noninvasive observation of root system growth adjusted for the subsequent root image acquisition with a reduced background noise. The method combines automated control of plant growth conditions with good imaging quality and high replicability of growth parameters

Arabinogalactan proteins are involved in root hair development in barley

The arabinogalactan proteins (AGPs) are involved in a range of plant processes, including cell differentiation and expansion. Here, barley root hair mutants and their wild-type parent cultivars were used, as a model system, to reveal the role of AGPs in root hair development. The treatment of roots with different concentrations of βGlcY (a reagent which binds to all classes of AGPs) inhibited or totally suppressed the development of root hairs in all of the cultivars. Three groups of AGP (recognized by the monoclonal antibodies LM2, LM14, and MAC207) were diversely localized in trichoblasts and atrichoblasts of root hair-producing plants. The relevant epitopes were present in wild-type trichoblast cell walls and cytoplasm, whereas in wild-type atrichoblasts and in all epidermal cells of a root hairless mutant, they were only present in the cytoplasm. In all of cultivars the higher expression of LM2, LM14, and MAC207 was observed in trichoblasts at an early stage of development. Additionally, the LM2 epitope was detected on the surface of primordia and root hair tubes in plants able to generate root hairs. The major conclusion was that the AGPs recognized by LM2, LM14, and MAC207 are involved in the differentiation of barley root epidermal cells, thereby implying a requirement for these AGPs for root hair development in barley

Asymmetric growth of root epidermal cells is related to the differentiation of root hair cells in Hordeum vulgare (L.)

The root epidermis of most vascular plants harbours two cell types, namely trichoblasts (capable of producing a root hair) and atrichoblasts. Here, in vivo analysis, confocal laser-scanning microscopy, transmission electron microscopy, histological analysis, and three-dimensional reconstruction were used to characterize the cell types present in the barley root epidermis and their distribution in the tissue. Both trichoblasts and atrichoblasts were present in the wildtype cultivars and could be distinguished from one another at an early stage. Trichoblast/atrichoblast differentiation depended on asymmetric cell expansion after a period of symmetrical cell division. After asymmetric growth, only the shorter epidermal cells could produce root hairs, whereas the longer cells became atrichoblasts. Moreover, the root epidermis did not develop root hairs at all if the epidermal cells did not differentiate into two asymmetric cell types. The root hairless phenotype of bald root barley (brb) and root hairless 1.b (rhl1.b) mutants was caused by a mutation in a gene related to the asymmetric expansion of the root epidermal cells. Additionally, the results showed that the mechanism of trichoblast/atrichoblast differentiation is not evolutionally conserved across the subfamilies of the Poaceae; in the Pooideae subfamily, both asymmetric division and asymmetric cell expansion have been observed

The role and regulation of ABI5 (ABA-Insensitive 5) in plant development, abiotic stress responses and phytohormone crosstalk

ABA Insensitive 5 (ABI5) is a basic leucine zipper transcription factor that plays a key role in the regulation of seed germination and early seedling growth in the presence of ABA and abiotic stresses. ABI5 functions in the core ABA signaling, which is composed of PYR/PYL/RCAR receptors, PP2C phosphatases and SnRK2 kinases, through the regulation of the expression of genes that contain the ABSCISIC ACID RESPONSE ELEMENT (ABRE) motif within their promoter region. The regulated targets include stress adaptation genes, e.g., LEA proteins. However, the expression and activation of ABI5 is not only dependent on the core ABA signaling. Many transcription factors such as ABI3, ABI4, MYB7 and WRKYs play either a positive or a negative role in the regulation of ABI5 expression. Additionally, the stability and activity of ABI5 are also regulated by other proteins through post-translational modifications such as phosphorylation, ubiquitination, sumoylation and S-nitrosylation. Moreover, ABI5 also acts as an ABA and other phytohormone signaling integrator. Components of auxin, cytokinin, gibberellic acid, jasmonate and brassinosteroid signaling and metabolism pathways were shown to take part in ABI5 regulation and/or to be regulated by ABI5. Monocot orthologs of AtABI5 have been identified. Although their roles in the molecular and physiological adaptations during abiotic stress have been elucidated, knowledge about their detailed action still remains elusive. Here, we describe the recent advances in understanding the action of ABI5 in early developmental processes and the adaptation of plants to unfavorable environmental conditions. We also focus on ABI5 relation to other phytohormones in the abiotic stress response of plants