SummaryIncreased tolerance of crops to low oxygen (hypoxia) during flooding is a key target for food security. In Arabidopsis thaliana (L.) Heynh., the N‐end rule pathway of targeted proteolysis controls plant responses to hypoxia by regulating the stability of group VII ethylene response factor (ERFVII) transcription factors, controlled by the oxidation status of amino terminal (Nt)‐cysteine (Cys). Here, we show that the barley (Hordeum vulgare L.) ERFVII BERF1 is a substrate of the N‐end rule pathway in vitro. Furthermore, we show that Nt‐Cys acts as a sensor for hypoxia in vivo, as the stability of the oxygen‐sensor reporter protein MCGGAIL‐GUS increased in waterlogged transgenic plants. Transgenic RNAi barley plants, with reduced expression of the N‐end rule pathway N‐recognin E3 ligase PROTEOLYSIS6 (HvPRT6), showed increased expression of hypoxia‐associated genes and altered seed germination phenotypes. In addition, in response to waterlogging, transgenic plants showed sustained biomass, enhanced yield, retention of chlorophyll, and enhanced induction of hypoxia‐related genes. HvPRT6 RNAi plants also showed reduced chlorophyll degradation in response to continued darkness, often associated with waterlogged conditions. Barley Targeting Induced Local Lesions IN Genomes (TILLING) lines, containing mutant alleles of HvPRT6, also showed increased expression of hypoxia‐related genes and phenotypes similar to RNAi lines. We conclude that the N‐end rule pathway represents an important target for plant breeding to enhance tolerance to waterlogging in barley and other cereals.

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.

Since the development of the Targeting Induced Local Lesions in Genome (TILLING) strategy, it has been applied in both plants and animals in many studies. The creation of an appropriate population is the first and most crucial step of TILLING. The goal is to obtain a highly mutagenized population that allows many mutations in any gene of interest to be found. Therefore, an effective method of mutation induction should be developed. A high mutation density is associated with saving time, costs, and the labor required for the development of a TILLING platform. The proper handling of the mutated generations, the establishment of a seed bank, and the development of a DNA library are essential for creating a TILLING population. The database in which all of the data from the molecular and phenotypic analyses are collected is a very useful tool for maintaining such population. Once developed, a TILLING population can serve as a renewable resource of mutations for research that uses both forward and reverse genetic approaches. In this chapter, we describe the methods for the development and maintenance of a TILLING population in barley.

Aluminum (Al) is one of the most important crust elements causing reduced plant production in acidic soils. Barley (Hordeum vulgare L.) is considered to be one of the crops that is most sensitive to Al, and the root cell wall is the primary target of Al toxicity. In this study, we evaluate the possible involvement of specific pectic epitopes in the cells of barley roots in response to aluminum exposure. We targeted four different pectic epitopes recognized by LM5, LM6, LM19, and LM20 antibodies using an immunocytochemical approach. Since Al becomes available and toxic to plants in acidic soils, we performed our analyses on barley roots that had been grown in acidic conditions (pH 4.0) with and without Al and in control conditions (pH 6.0). Differences connected with the presence and distribution of the pectic epitopes between the control and Al-treated roots were observed. In the Al-treated roots, pectins with galactan sidechains were detected with a visually lower fluorescence intensity than in the control roots while pectins with arabinan sidechains were abundantly present. Furthermore, esterified homogalacturonans (HGs) were present with a visually higher fluorescence intensity compared to the control, while methyl-esterified HGs were present in a similar amount. Based on the presented results, it was concluded that methyl-esterified HG can be a marker for newly arising cell walls. Additionally, histological changes were detected in the roots grown under Al exposure. Among them, an increase in root diameter, shortening of root cap, and increase in the size of rhizodermal cells and divisions of exodermal and cortex cells were observed. The presented data extend upon the knowledge on the chemical composition of the cell wall of barley root cells under stress conditions. The response of cells to Al can be expressed by the specific distribution of pectins in the cell wall and, thus, enables the knowledge on Al toxicity to be extended by explaining the mechanism by which Al inhibits root elongation.

Ataxia Telangiectasia and Rad-3-related protein (ATR) is a DNA damage signaling kinase required for the monitoring of DNA integrity. Together with ATM and SOG1, it is a key player in the transcriptional regulation of DNA damage response (DDR) genes in plants. In this study, we describe the role of ATR in the DDR pathway in barley and the function of the HvATR gene in response to DNA damages induced by aluminum toxicity. Aluminum is the third most abundant element in the Earth’s crust. It becomes highly phytotoxic in acidic soils, which comprise more than 50% of arable lands worldwide. At low pH, Al is known to be a genotoxic agent causing DNA damage and cell cycle arrest. We present barley mutants, hvatr.g and hvatr.i, developed by TILLING strategy. The hvatr.g mutant carries a G6054A missense mutation in the ATR gene, leading to the substitution of a highly conserved amino acid in the protein (G1015S). The hvatr.g mutant showed the impaired DDR pathway. It accumulated DNA damages in the nuclei of root meristem cells when grown in control conditions. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis revealed that 60% of mutant nuclei possessed DNA nicks and breaks, whereas in the wild type only 2% of the nuclei were TUNEL-positive. The high frequency of DNA damages did not lead to the inhibition of the cell cycle progression, but the mutant showed an increased number of cells in the G2/M phase. In response to treatments with different Al doses, hvatr.g showed a high level of tolerance. The retention of root growth, which is the most evident symptom of Al toxicity, was not observed in the mutant, as it was in its parent variety. Furthermore, Al treatment increased the level of DNA damages, but did not affect the mitotic activity and the cell cycle profile in the hvatr.g mutant. A similar phenotype was observed for the hvatr.i mutant, carrying another missense mutation leading to G903E substitution in the HvATR protein. Our results demonstrate that the impaired mechanism of DNA damage response may lead to aluminum tolerance. They shed a new light on the role of the ATR-dependent DDR pathway in an agronomically important species.