Recent advances in large-scale genome sequencing projects have opened up new possibilities for the application of conventional mutation techniques in not only forward but also reverse genetics strategies. TILLING (Targeting Induced Local Lesions IN Genomes) was developed a decade ago as an alternative to insertional mutagenesis. It takes advantage of classical mutagenesis, sequence availability and high-throughput screening for nucleotide polymorphisms in a targeted sequence. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of its genome size and ploidy level. The TILLING protocol provides a high frequency of point mutations distributed randomly in the genome. The great mutagenic potential of chemical agents to generate a high rate of nucleotide substitutions has been proven by the high density of mutations reported for TILLING populations in various plant species. For most of them, the analysis of several genes revealed 1 mutation/200–500 kb screened and much higher densities were observed for polyploid species, such as wheat. High-throughput TILLING permits the rapid and low-cost discovery of new alleles that are induced in plants. Several research centres have established a TILLING public service for various plant species. The recent trends in TILLING procedures rely on the diversification of bioinformatic tools, new methods of mutation detection, including mismatch-specific and sensitive endonucleases, but also various alternatives for LI-COR screening and single nucleotide polymorphism (SNP) discovery using next-generation sequencing technologies. The TILLING strategy has found numerous applications in functional genomics. Additionally, wide applications of this throughput method in basic and applied research have already been implemented through modifications of the original TILLING strategy, such as Ecotilling or Deletion TILLING.

Reduced plant height and culm robustness are quantitative characteristics important for assuring cereal crop yield and quality under adverse weather conditions. A very limited number of short-culm mutant alleles were introduced into commercial crop cultivars during the Green Revolution. We identified phenotypic traits, including sturdy culm, specific for deficiencies in brassinosteroid biosynthesis and signaling in semidwarf mutants of barley (Hordeum vulgare). This set of characteristic traits was explored to perform a phenotypic screen of near-isogenic short-culm mutant lines from the brachytic, breviaristatum, dense spike, erectoides, semibrachytic, semidwarf, and slender dwarf mutant groups. In silico mapping of brassinosteroid-related genes in the barley genome in combination with sequencing of barley mutant lines assigned more than 20 historic mutants to three brassinosteroid-biosynthesis genes (BRASSINOSTEROID-6-OXIDASE, CONSTITUTIVE PHOTOMORPHOGENIC DWARF, and DIMINUTO) and one brassinosteroid-signaling gene (BRASSINOSTEROID-INSENSITIVE1 [HvBRI1]). Analyses of F2 and M2 populations, allelic crosses, and modeling of nonsynonymous amino acid exchanges in protein crystal structures gave a further understanding of the control of barley plant architecture and sturdiness by brassinosteroid-related genes. Alternatives to the widely used but highly temperature-sensitive uzu1.a allele of HvBRI1 represent potential genetic building blocks for breeding strategies with sturdy and climate-tolerant barley cultivars.

Strigolactones (SL) are the youngest group of plant hormones responsible for shaping plant architecture, especially the branching of shoots. However, recent studies provided new insights into the functioning of SL, confirming their participation in regulating the plant response to various types of abiotic stresses, including water deficit, soil salinity and osmotic stress. On the other hand, abscisic acid (ABA), commonly referred as a stress hormone, is the molecule that crucially controls the plant response to adverse environmental conditions. Since the SL and ABA share a common precursor in their biosynthetic pathways, the interaction between both phytohormones has been largely studied in the literature. Under optimal growth conditions, the balance between ABA and SL content is maintained to ensure proper plant development. At the same time, the water deficit tends to inhibit SL accumulation in the roots, which serves as a sensing mechanism for drought, and empowers the ABA production, which is necessary for plant defense responses. The SL-ABA cross-talk at the signaling level, especially regarding the closing of the stomata under drought conditions, still remains poorly understood. Enhanced SL content in shoots is likely to stimulate the plant sensitivity to ABA, thus reducing the stomatal conductance and improving the plant survival rate. Besides, it was proposed that SL might promote the closing of stomata in an ABA-independent way. Here, we summarize the current knowledge regarding the SL and ABA interactions by providing new insights into the function, perception and regulation of both phytohormones during abiotic stress response of plants, as well as revealing the gaps in the current knowledge of SL-ABA cross-talk.

Strigolactones (SLs) are a new group of plant hormones, which have been intensively investigated during the last few years. The wide spectrum of SLs actions, including the regulation of shoot/root architecture, and the stimulation of the interactions between roots and fungi or bacteria, as well as the stimulation of germination of parasitic plants, indicates that this group of hormones may play an important role in the mechanisms that control soil exploration, and the root-mediated uptake of nutrients. Current studies have shown that SLs might be factors that have an influence on the plant response to a deficiency of macronutrients. Experimental data from the last four years have confirmed that the biosynthesis and exudation of SLs are increased under phosphorus and nitrogen deficiency. All these data suggest that SLs may regulate the complex response to nutrient stress, which include not only the modification of the plant developmental process, but also the cooperation with other organisms in order to minimize the effects of threats. In this paper the results of studies that indicate that SLs play an important role in the response to nutrient stress are reviewed and the consequences of the higher biosynthesis and exudation of SLs in response to phosphorus and nitrogen deficiency are discussed.

TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN₃) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M₂ plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M₃ progeny of 3,481 M₂ individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M₂ plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.

Strigolactones were described as a new group of phytohormones in 2008 and since then notable large number of their functions has been uncovered, including the regulation of plant growth and development, interactions with other organisms and a plant’s response to different abiotic stresses. In the last year, investigations of the strigolactone biosynthesis pathway in two model species, Arabidopsis thaliana and Oryza sativa, resulted in great progress in understanding the functions of four enzymes that are involved in this process. We performed in silico analyses, including the identification of the cis-regulatory elements in the promoters of genes encoding proteins of the strigolactone biosynthesis pathway and the identification of the miRNAs that are able to regulate their posttranscriptional level. We also searched the databases that contain the microarray data for the genes that were analyzed from both species in order to check their expression level under different growth conditions. The results that were obtained indicate that there are universal regulations of expression of all of the genes that are involved in the strigolactone biosynthesis in Arabidopsis and rice, but on the other hand each stage of strigolactone production may be additionally regulated independently. This work indicates the presence of crosstalk between strigolactones and almost all of the other phytohormones and suggests the role of strigolactones in the response to abiotic stresses, such as wounding, cold or flooding, as well as in the response to biotic stresses.