Search

One of the main signal transduction pathways that modulate plant growth and stress responses, including drought, is the action of phytohormones. Recent advances in omics approaches have facilitated the exploration of plant genomes. However, the molecular mechanisms underlying the response in the crown of barley, which plays an essential role in plant performance under stress conditions and regeneration after stress treatment, remain largely unclear. The objective of the present study was the elucidation of drought-induced molecular reactions in the crowns of different barley phytohormone mutants. We verified the hypothesis that defects of gibberellins, brassinosteroids, and strigolactones action affect the transcriptomic, proteomic, and hormonal response of barley crown to the transitory drought influencing plant development under stress. Moreover, we assumed that due to the strong connection between strigolactones and branching the hvdwarf14.d mutant, with dysfunctional receptor of strigolactones, manifests the most abundant alternations in crowns and phenotype under drought. Finally, we expected to identify components underlying the core response to drought which are independent of the genetic background. Large-scale analyses were conducted using gibberellins-biosynthesis, brassinosteroids-signaling, and strigolactones-signaling mutants, as well as reference genotypes. Detailed phenotypic evaluation was also conducted. The obtained results clearly demonstrated that hormonal disorders caused by mutations in the HvGA20ox2, HvBRI1, and HvD14 genes affected the multifaceted reaction of crowns to drought, although the expression of these genes was not induced by stress. The study further detected not only genes and proteins that were involved in the drought response and reacted specifically in mutants compared to the reaction of reference genotypes and vice versa, but also the candidates that may underlie the genotype-universal stress response. Furthermore, candidate genes involved in phytohormonal interactions during the drought response were identified. We also found that the interplay between hormones, especially gibberellins and auxins, as well as strigolactones and cytokinins may be associated with the regulation of branching in crowns exposed to drought. Overall, the present study provides novel insights into the molecular drought-induced responses that occur in barley crowns.

Since its introduction in 2000, the TILLING strategy has been widely used in plant research to create novel genetic diversity. TILLING is based on chemical or physical mutagenesis followed by the rapid identification of mutations within genes of interest. TILLING mutants may be used for functional analysis of genes and being nontransgenic, they may be directly used in pre-breeding programs. Nevertheless, classical mutagenesis is a random process, giving rise to mutations all over the genome. Therefore TILLING mutants carry background mutations, some of which may affect the phenotype and should be eliminated, which is often time-consuming. Recently, new strategies of targeted genome editing, including CRISPR/Cas9-based methods, have been developed and optimized for many plant species. These methods precisely target only genes of interest and produce very few off-targets. Thus, the question arises: is it the end of TILLING era in plant studies? In this review, we recap the basics of the TILLING strategy, summarize the current status of plant TILLING research and present recent TILLING achievements. Based on these reports, we conclude that TILLING still plays an important role in plant research as a valuable tool for generating genetic variation for genomics and breeding projects.

Abstract Background High temperature shock is becoming increasingly common in our climate, affecting plant growth and productivity. The ability of a plant to survive stress is a complex phenomenon. One of the essential tissues for plant performance under various environmental stimuli is the crown. However, the molecular characterization of this region remains poorly investigated. Gibberellins play a fundamental role in whole-plant stature formation. This study identified plant stature modifications and crown-specific transcriptome re-modeling in gibberellin-deficient barley sdw1.a (BW827) and sdw1.d (BW828) mutants exposed to increased temperature. Results The deletion around the sdw1 gene in BW827 was found to encompass at least 13 genes with primarily regulatory functions. A bigger genetic polymorphism of BW828 than of BW827 in relation to wild type was revealed. Transcriptome-wide sequencing (RNA-seq) revealed several differentially expressed genes involved in gibberellin metabolism and heat response located outside of introgression regions. It was found that HvGA20ox4, a paralogue of the HvGA20ox2 gene, was upregulated in BW828 relative to other genotypes, which manifested as basal internode elongation. The transcriptome response to elevated temperature differed in the crown of sdw1.a and sdw1.d mutants; it was most contrasting for HvHsf genes upregulated under elevated temperature in BW828, whereas those specific to BW827 were downregulated. In-depth examination of sdw1 mutants revealed also some differences in their phenotypes and physiology. Conclusions We concluded that despite the studied sdw1 mutants being genetically related, their heat response seemed to be genotype-specific and observed differences resulted from genetic background diversity rather than single gene mutation, multiple gene deletion, or allele-specific expression of the HvGA20ox2 gene. Differences in the expressional reaction of genes to heat in different sdw1 mutants, found to be independent of the polymorphism, could be further explained by in-depth studies of the regulatory factors acting in the studied system. Our findings are particularly important in genetic research area since molecular response of crown tissue has been marginally investigated, and can be useful for wide genetic research of crops since barley has become a model plant for them.

Diverse processes leading to doubled haploid (DH) plant production, such as microspore embryogenesis, gynogenesis, and distant hybridization followed by genome elimination, are based on the unique ability of plant cells to form haploid embryos without fertilization. All of these are possible because of various in vitro culture systems that enable the growth and development of tissues or single cells outside of the parental organism. The possibility of re-directing cell development from its original pathway to embryogenesis brings several benefits to many research areas, but the most important is the possibility of its implementation in breeding programs. This review summarizes the achievements of Polish research groups in studies of the mechanisms of haploid/DH embryo development and demonstrates the practical applications of these systems in basic studies and plant breeding. It shows the results of studies on economically important crops including barley (Hordeum vulgare L.), oilseed rape (Brassica napus L.), triticale (×Triticosecale Wittm.), oat (Avena sativa L.), rye (Secale cereale L.), sugar beet (Beta vulgaris ssp. vulgaris L.), and some vegetable species, including carrot (Daucus carota L.), onion (Allium cepa L.), red beet (Beta vulgaris L.), and members of the Brassicaceae.

Abstract Background Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley. Results We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, cv. ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNA Glu , which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants. Conclusions Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoints of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanisms underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

Improvements to massively parallel sequencing have allowed the routine recovery of natural and induced sequence variants. A broad range of biological disciplines have benefited from this, ranging from plant breeding to cancer research. The need for high sequence coverage to accurately recover single nucleotide variants and small insertions and deletions limits the applicability of whole genome approaches. This is especially true in organisms with a large genome size or for applications requiring the screening of thousands of individuals, such as the reverse-genetic technique known as TILLING. Using PCR to target and sequence chosen genomic regions provides an attractive alternative as the vast reduction in interrogated bases means that sample size can be dramatically increased through amplicon multiplexing and multi-dimensional sample pooling while maintaining suitable coverage for recovery of small mutations. Direct sequencing of PCR products is limited, however, due to limitations in read lengths of many next generation sequencers. In the present study we show the optimization and use of ultrasonication for the simultaneous fragmentation of multiplexed PCR amplicons for TILLING highly pooled samples. Sequencing performance was evaluated in a total of 32 pooled PCR products produced from 4096 chemically mutagenized Hordeum vulgare DNAs pooled in three dimensions. Evaluation of read coverage and base quality across amplicons suggests this approach is suitable for high-throughput TILLING and other applications employing highly pooled complex sampling schemes. Induced mutations previously identified in a traditional TILLING screen were recovered in this dataset further supporting the efficacy of the approach.

Aluminum (Al) toxicity is considered to be the most harmful abiotic stress in acidic soils that today comprise more than 50% of the world’s arable lands. Barley belongs to a group of crops that are most sensitive to Al in low pH soils. We present the RNA-seq analysis of root meristems of barley seedlings grown in hydroponics at optimal pH (6.0), low pH (4.0), and low pH with Al (10 μM of bioavailable Al3+ ions). Two independent experiments were conducted: with short-term (24 h) and long-term (7 days) Al treatment. In the short-term experiment, more genes were differentially expressed (DEGs) between root meristems grown at pH = 6.0 and pH = 4.0, than between those grown at pH = 4.0 with and without Al treatment. The genes upregulated by low pH were associated mainly with response to oxidative stress, cell wall organization, and iron ion binding. Among genes upregulated by Al, overrepresented were those related to response to stress condition and calcium ion binding. In the long-term experiment, the number of DEGs between hydroponics at pH = 4.0 and 6.0 were lower than in the short-term experiment, which suggests that plants partially adapted to the low pH. Interestingly, 7 days Al treatment caused massive changes in the transcriptome profile. Over 4,000 genes were upregulated and almost 2,000 genes were downregulated by long-term Al stress. These DEGs were related to stress response, cell wall development and metal ion transport. Based on our results we can assume that both, Al3+ ions and low pH are harmful to barley plants. Additionally, we phenotyped the root system of barley seedlings grown in the same hydroponic conditions for 7 days at pH = 6.0, pH = 4.0, and pH = 4.0 with Al. The results correspond to transcriptomic data and show that low pH itself is a stress factor that causes a significant reduction of root growth and the addition of aluminum further increases this reduction. It should be noted that in acidic arable lands, plants are exposed simultaneously to both of these stresses. The presented transcriptome analysis may help to find potential targets for breeding barley plants that are more tolerant to such conditions.

ABA INSENSITIVE 5 (ABI5) is a basic leucine zipper (bZIP) transcription factor which acts in the abscisic acid (ABA) network and is activated in response to abiotic stresses. However, the precise role of barley (Hordeum vulgare) ABI5 in ABA signaling and its function under stress remains elusive. Here, we show that HvABI5 is involved in ABA-dependent regulation of barley response to drought stress. We identified barley TILLING mutants carrying different alleles in the HvABI5 gene and we studied in detail the physiological and molecular response to drought and ABA for one of them. The hvabi5.d mutant, carrying G1751A transition, was insensitive to ABA during seed germination, yet it showed the ability to store more water than its parent cv. “Sebastian” (WT) in response to drought stress. The drought-tolerant phenotype of hvabi5.d was associated with better membrane protection, higher flavonoid content, and faster stomatal closure in the mutant under stress compared to the WT. The microarray transcriptome analysis revealed up-regulation of genes associated with cell protection mechanisms in the mutant. Furthermore, HvABI5 target genes: HVA1 and HVA22 showed higher activity after drought, which may imply better adaptation of hvabi5.d to stress. On the other hand, chlorophyll content in hvabi5.d was lower than in WT, which was associated with decreased photosynthesis efficiency observed in the mutant after drought treatment. To verify that HvABI5 acts in the ABA-dependent manner we analyzed expression of selected genes related to ABA pathway in hvabi5.d and its WT parent after drought and ABA treatments. The expression of key genes involved in ABA metabolism and signaling differed in the mutant and the WT under stress. Drought-induced increase of expression of HvNCED1, HvBG8, HvSnRK2.1, and HvPP2C4 genes was 2–20 times higher in hvabi5.d compared to “Sebastian”. We also observed a faster stomatal closure in hvabi5.d and much higher induction of HvNCED1 and HvSnRK2.1 genes after ABA treatment. Together, these findings demonstrate that HvABI5 plays a role in regulation of drought response in barley and suggest that HvABI5 might be engaged in the fine tuning of ABA signaling by a feedback regulation between biosynthetic and signaling events. In addition, they point to different mechanisms of HvABI5 action in regulating drought response and seed germination in barley.

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.

Meiosis is a process of essential importance for sexual reproduction, as it leads to production of gametes. The recombination event (crossing-over) generates genetic variation by introducing new combination of alleles. The first step of crossing-over is introduction of a targeted double-strand break (DSB) in DNA. DMC1 (Disrupted Meiotic cDNA1) is a recombinase that is specific only for cells undergoing meiosis and takes part in repair of such DSBs by searching and invading homologous sequences that are subsequently used as a template for the repair process. Although role of the DMC1 gene has been validated in Arabidopsis thaliana, a functional analysis of its homolog in barley, a crop species of significant importance in agriculture, has never been performed. Here, we describe the identification of barley mutants carrying substitutions in the HvDMC1 gene. We performed mutational screening using TILLING (Targeting Induced Local Lesions IN Genomes) strategy and the barley TILLING population, HorTILLUS, developed after double-treatment of spring barley cultivar ‘Sebastian’ with sodium azide and N-methyl-N-nitrosourea. One of the identified alleles, dmc1.c, was found independently in two different M2 plants. The G2571A mutation identified in this allele leads to a substitution of the highly conserved amino acid (arginine-183 to lysine) in the DMC1 protein sequence. Two mutant lines carrying the same dmc1.c allele show similar disturbances during meiosis. The chromosomal aberrations included anaphase bridges and chromosome fragments in anaphase/telophase I and anaphase/telophase II, as well as micronuclei in tetrads. Moreover, atypical tetrads containing three or five cells were observed. A highly increased frequency of all chromosome aberrations during meiosis have been observed in the dmc1.c mutants compared to parental variety. The results indicated that DMC1 is required for the DSB repair, crossing-over and proper chromosome disjunction during meiosis in barley.

The core abscisic acid (ABA) signaling pathway consists of receptors, phosphatases, kinases and transcription factors, among them ABA INSENSITIVE 5 (ABI5) and ABRE BINDING FACTORs/ABRE-BINDING PROTEINs (ABFs/AREBs), which belong to the BASIC LEUCINE ZIPPER (bZIP) family and control expression of stress-responsive genes. ABI5 is mostly active in seeds and prevents germination and post-germinative growth under unfavorable conditions. The activity of ABI5 is controlled at transcriptional and protein levels, depending on numerous regulators, including components of other phytohormonal pathways. ABFs/AREBs act redundantly in regulating genes that control physiological processes in response to stress during vegetative growth. In this review, we focus on recent reports regarding ABI5 and ABFs/AREBs functions during abiotic stress responses, which seem to be partially overlapping and not restricted to one developmental stage in Arabidopsis and other species. Moreover, we point out that ABI5 and ABFs/AREBs play a crucial role in the core ABA pathway’s feedback regulation. In this review, we also discuss increased stress tolerance of transgenic plants overexpressing genes encoding ABA-dependent bZIPs. Taken together, we show that ABI5 and ABFs/AREBs are crucial ABA-dependent transcription factors regulating processes essential for plant adaptation to stress at different developmental stages.

Tonoplast Intrinsic Proteins (TIP) are plant aquaporins that are primarily localized in the tonoplast and play a role in the bidirectional flux of water and other substrates across a membrane. In barley, eleven members of the HvTIP gene subfamily have been identified. Here, we describe the transcription profile of the HvTIP genes in the leaves of barley seedlings being grown under optimal moisture conditions, drought stress and a re-watering phase. The applied drought stress caused a 55% decrease in the relative water content (RWC) in seedlings, while re-watering increased the RWC to 90% of the control. Our analysis showed that all HvTIP genes, except HvTIP3;2, HvTIP4;3 and HvTIP5.1, were expressed in leaves of ten-day-old barley seedlings under optimal water conditions with the transcripts of HvTIP2;3, HvTIP1;2 and HvTIP1;1 being the most abundant. We showed, for the first time in barley, a significant variation in the transcriptional activity between the analysed genes under drought stress. After drought treatment, five HvTIP genes, which are engaged in water transport, were down-regulated to varying degrees, while two, HvTIP3;1 and HvTIP4;1, were up-regulated. The HvTIP3;1 isoform, which is postulated as transporting hydrogen peroxide, expressed the highest increase of activity (ca. 5000x) under drought stress, thus indicating its importance in the response to this stress. Re-hydration caused the return of the expression of many genes to the level that was observed under optimal moisture conditions or, at least, a change in this direction Additionally, we examined the promotor regions of HvTIP and detected the presence of the cis-regulatory elements that are connected with the hormone and stress responses in all of the genes. Overall, our results suggest that 7 of 11 studied HvTIP (HvTIP1;1, HvTIP1;2, HvTIP2;1, HvTIP2;2, HvTIP2;3, HvTIP3;1, HvTIP4;1) have an important function during the adaptation of barley to drought stress conditions. We discuss the identified drought-responsive HvTIP in terms of their function in the adaptation of barley to this stress.

Root hairs are the part of root architecture contributing significantly to the root surface area. Their role is particularly substantial in maintaining plant growth under stress conditions, however, knowledge on mechanism of root hair differentiation is still limited for majority of crop species, including barley. Here, we report the results of a map-based identification of a candidate gene responsible for the lack of root epidermal cell differentiation, which results in the lack of root hairs in barley. The analysis was based on the root hairless barley mutant rhl1.b, obtained after chemical mutagenesis of spring cultivar ‘Karat’. The rhl1 gene was located in chromosome 7HS in our previous studies. Fine mapping allowed to narrow the interval encompassing rhl1 gene to 3.7 cM, which on physical barley map spans a region of 577 kb. Five high confidence genes are located within this region and their sequencing resulted in the identification of A>T mutation in one candidate, HORVU7Hr1G030250 (MLOC_38567), differing the mutant from its parent variety. The mutation, located in the 3′ splice-junction site, caused the retention of the last intron, 98 bp long, in mRNA of rhl1.b allele. This resulted in the frameshift, the synthesis of 71 abnormal amino acids and introduction of premature STOP codon in mRNA. The mutation was present in the recombinants from the mapping population (F2rhl1.b × ‘Morex’) that lacked root hairs. The candidate gene encodes a bHLH transcription factor with LRL domain and may be involved in early stages of root hair cell development. We discuss the possible involvement of HORVU7Hr1G030250 in this process, as the best candidate responsible for early stages of rhizodermis differentiation in barley.

Drought is one of the most adverse abiotic factors limiting growth and productivity of crops. Among them is barley, ranked fourth cereal worldwide in terms of harvested acreage and production. Plants have evolved various mechanisms to cope with water deficit at different biological levels, but there is an enormous challenge to decipher genes responsible for particular complex phenotypic traits, in order to develop drought tolerant crops. This work presents a comprehensive approach for elucidation of molecular mechanisms of drought tolerance in barley at the seedling stage of development. The study includes mapping of QTLs for physiological and biochemical traits associated with drought tolerance on a high-density function map, projection of QTL confidence intervals on barley physical map, and the retrievement of positional candidate genes (CGs), followed by their prioritization based on Gene Ontology (GO) enrichment analysis. A total of 64 QTLs for 25 physiological and biochemical traits that describe plant water status, photosynthetic efficiency, osmoprotectant and hormone content, as well as antioxidant activity, were positioned on a consensus map, constructed using RIL populations developed from the crosses between European and Syrian genotypes. The map contained a total of 875 SNP, SSR and CGs, spanning 941.86 cM with resolution of 1.1 cM. For the first time, QTLs for ethylene, glucose, sucrose, maltose, raffinose, α-tocopherol, γ-tocotrienol content, and catalase activity, have been mapped in barley. Based on overlapping confidence intervals of QTLs, 11 hotspots were identified that enclosed more than 60% of mapped QTLs. Genetic and physical map integration allowed the identification of 1,101 positional CGs within the confidence intervals of drought response-specific QTLs. Prioritization resulted in the designation of 143 CGs, among them were genes encoding antioxidants, carboxylic acid biosynthesis enzymes, heat shock proteins, small auxin up-regulated RNAs, nitric oxide synthase, ATP sulfurylases, and proteins involved in regulation of flowering time. This global approach may be proposed for identification of new CGs that underlies QTLs responsible for complex traits.

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.

Plant survival in adverse environmental conditions requires a substantial change in the metabolism, which is reflected by the extensive transcriptome rebuilding upon the occurrence of the stress. Therefore, transcriptomic studies offer an insight into the mechanisms of plant stress responses. Here, we present the results of global gene expression profiling of roots and leaves of two barley genotypes with contrasting ability to cope with drought stress. Our analysis suggests that drought tolerance results from a certain level of transcription of stress-influenced genes that is present even before the onset of drought. Genes that predispose the plant to better drought survival play a role in the regulatory network of gene expression, including several transcription factors, translation regulators and structural components of ribosomes. An important group of genes is involved in signaling mechanisms, with significant contribution of hormone signaling pathways and an interplay between ABA, auxin, ethylene and brassinosteroid homeostasis. Signal transduction in a drought tolerant genotype may be more efficient through the expression of genes required for environmental sensing that are active already during normal water availability and are related to actin filaments and LIM domain proteins, which may function as osmotic biosensors. Better survival of drought may also be attributed to more effective processes of energy generation and more efficient chloroplasts biogenesis. Interestingly, our data suggest that several genes involved in a photosynthesis process are required for the establishment of effective drought response not only in leaves, but also in roots of barley. Thus, we propose a hypothesis that root plastids may turn into the anti-oxidative centers protecting root macromolecules from oxidative damage during drought stress. Specific genes and their potential role in building up a drought-tolerant barley phenotype is extensively discussed with special emphasis on processes that take place in barley roots. When possible, the interconnections between particular factors are emphasized to draw a broader picture of the molecular mechanisms of drought tolerance in barley.

Barley is one of the cereals that are most sensitive to aluminum (Al). Al in acid soils limits barley growth and development and, as a result, its productivity. The inhibition of root growth is a widely accepted indicator of Al stress. Al toxicity is affected by many factors including the culture medium, pH, Al concentration and the duration of the treatment. However, Al can act differently in different species and still Al toxicity in barley deserves study. Since the mechanism of Al toxicity is discussed we cytogenetically describe the effects of different doses of bioavailable Al on the barley nuclear genome-mitotic activity, cell cycle profile and DNA integrity. At the same time, we tested an established deep-water culture (DWC) hydroponics system and analyzed the effects of Al on the root system parameters using WinRHIZO software. We demonstrated the cytotoxic and genotoxic effect of Al in barley root cells. We showed that Al treatment significantly reduced the mitotic activity of the root tip cells and it also induced micronuclei and damaged nuclei. The DNA-damaging effect of Al was observed using the TUNEL test. We define the inhibitory influence of Al on DNA replication in barley. Analysis with the labelling and detection of 5-ethynyl-2'-deoxyuridin (EdU) showed that the treatment with Al significantly decreased the frequency of S phase cells. We also demonstrated that Al exposure led to changes in the cell cycle profile of barley root tips. The delay of cell divisions observed as increased frequency of cells in G2/M phase after Al treatment was reported using flow cytometry.

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop plants with large and complex genomes.

CBP20 (Cap-Binding Protein 20) encodes a small subunit of the cap-binding complex (CBC), which is involved in the conserved cell processes related to RNA metabolism in plants and, simultaneously, engaged in the signaling network of drought response, which is dependent on ABA. Here, we report the enhanced tolerance to drought stress of barley mutant in the HvCBP20 gene manifested at the morphological, physiological, and transcriptomic levels. Physiological analyses revealed differences between the hvcbp20.ab mutant and its WT in response to a water deficiency. The mutant exhibited a higher relative water content (RWC), a lower stomatal conductance and changed epidermal pattern compared to the WT after drought stress. Transcriptome analysis using the Agilent Barley Microarray integrated with observed phenotypic traits allowed to conclude that the hvcbp20.ab mutant exhibited better fitness to stress conditions by its much more efficient and earlier activation of stress-preventing mechanisms. The network hubs involved in the adjustment of hvcbp20.ab mutant to the drought conditions were proposed. These results enabled to make a significant progress in understanding the role of CBP20 in the drought stress response.

The rhizosphere, the thin layer of soil surrounding and influenced by plant roots, defines a distinct and selective microbial habitat compared to unplanted soil. The microbial communities inhabiting the rhizosphere, the rhizosphere microbiota, engage in interactions with their host plants which span from parasitism to mutualism. Therefore, the rhizosphere microbiota emerges as one of the determinants of yield potential in crops. Studies conducted with different plant species have unequivocally pointed to the host plant as a driver of the microbiota thriving at the root–soil interface. Thus far, the host genetic traits shaping the rhizosphere microbiota are not completely understood. As root hairs play a critical role in resource exchanges between plants and the rhizosphere, we hypothesized that they can act as a determinant of the microbiota thriving at the root–soil interface. To test this hypothesis, we took advantage of barley (Hordeum vulgare) mutant lines contrasting for their root hair characteristics. Plants were grown in two agricultural soils, differentiating in their organic matter contents, under controlled environmental conditions. At early stem elongation rhizosphere specimens were collected and subjected to high-resolution 16S rRNA gene profiling. Our data revealed that the barley rhizosphere microbiota is largely dominated by members of the phyla Bacteroidetes and Proteobacteria, regardless of the soil type and the root hair characteristics of the host plant. Conversely, ecological indices calculated using operational taxonomic units (OTUs) presence, abundance, and phylogeny revealed a significant impact of root hair mutations on the composition of the rhizosphere microbiota. In particular, our data indicate that mutant plants host a reduced-complexity community compared to wild-type genotypes and unplanted soil controls. Congruently, the host genotype explained up to 18% of the variation in ecological distances computed for the rhizosphere samples. Importantly, this effect is manifested in a soil-dependent manner. A closer inspection of the sequencing profiles revealed that the root hair-dependent diversification of the microbiota is supported by a taxonomically narrow group of bacteria, with a bias for members of the orders Actinomycetales, Burkholderiales, Rhizobiales, Sphingomonadales, and Xanthomonadales. Taken together, our results indicate that the presence and function of root hairs are a determinant of the bacterial community thriving in the rhizosphere and their perturbations can markedly impact on the recruitment of individual members of the microbiota.

Brassinosteroids (BRs) are a class of steroid phytohormones, which regulate various processes of morphogenesis and physiology – from seed development to regulation of flowering and senescence. An accumulating body of evidence indicates that BRs take part in regulation of physiological reactions to various stress conditions, including drought. Many of the physiological functions of BRs are regulated by a complicated, and not fully elucidated network of interactions with metabolic pathways of other phytohormones. Therefore, the aim of this study was to characterize phytohormonal homeostasis in barley (Hordeum vulgare) in reaction to drought and validate role of BRs in regulation of this process. Material of this study included the barley cultivar ‘Bowman’ and five Near-Isogenic Lines (NILs) representing characterized semi-dwarf mutants of several genes encoding enzymes participating in BR biosynthesis and signaling. Analysis of endogenous BRs concentrations in these NILs confirmed that their phenotypes result from abnormalities in BR metabolism. In general, concentrations of eighteen compounds, representing various classes of phytohormones, including brassinosteroids, auxins, cytokinins, gibberellins, abscisic acid, salicylic acid and jasmonic acid were analyzed under control and drought conditions in the ‘Bowman’ cultivar and the BR-deficient NILs. Drought induced a significant increase in accumulation of the biologically active form of BRs – castasterone in all analyzed genotypes. Another biologically active form of BRs – 24-epi-brassinolide - was identified in one, BR-insensitive NIL under normal condition, but its accumulation was drought-induced in all analyzed genotypes. Analysis of concentration profiles of several compounds representing gibberellins allowed an insight into the BR-dependent regulation of gibberellin biosynthesis. The concentration of the gibberellic acid GA7 was significantly lower in all NILs when compared with the ‘Bowman’ cultivar, indicating that GA7 biosynthesis represents an enzymatic step at which the stimulating effect of BRs on gibberellin biosynthesis occurs. Moreover, the accumulation of GA7 is significantly induced by drought in all the genotypes. Biosynthesis of jasmonic acid is also a BR-dependent process, as all the NILs accumulated much lower concentrations of this hormone when compared with the ‘Bowman’ cultivar under normal condition, however the accumulation of jasmonic acid, abscisic acid and salicylic acid were significantly stimulated by drought.

ABA Insensitive 5 (ABI5) is a basic leucine zipper (bZIP) 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 orthologues of AtABI5 have been identified. Although their role 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.

In response to climatic changes, breeding programmes should be aimed at creating new cultivars with improved resistance to water scarcity. The objective of this study was to examine the yield potential of barley recombinant inbred lines (RILs) derived from three cross-combinations of European and Syrian spring cultivars, and to identify quantitative trait loci (QTLs) for yield-related traits in these populations. RILs were evaluated in field experiments over a period of three years (2011 to 2013) and genotyped with simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers; a genetic map for each population was constructed and then one consensus map was developed. Biological interpretation of identified QTLs was achieved by reference to Ensembl Plants barley gene space. Twelve regions in the genomes of studied RILs were distinguished after QTL analysis. Most of the QTLs were identified on the 2H chromosome, which was the hotspot region in all three populations. Syrian parental cultivars contributed alleles decreasing traits' values at majority of QTLs for grain weight, grain number, spike length and time to heading, and numerous alleles increasing stem length. The phenomic and molecular approaches distinguished the lines with an acceptable grain yield potential combining desirable features or alleles from their parents, that is, early heading from the Syrian breeding line (Cam/B1/CI08887//CI05761) and short plant stature from the European semidwarf cultivar (Maresi).