The improvement of the in vitro plant regeneration in barley with the epigenetic modifier of histone acetylation, trichostatin A.

Genotype-limited plant regeneration is one of the main obstacles to the broader use of genetic transformation in barley breeding. Thus, developing new approaches that might improve responses of in vitro recalcitrant genotypes remains at the center of barley biotechnology. Here, we analyzed different barley genotypes, including "Golden Promise," a genotype commonly used in the genetic transformation, and four malting barley cultivars of poor regenerative potential. The expression of hormone-related transcription factor (TF) genes with documented roles in plant regeneration was analyzed in genotypes with various plant-regenerating capacities. The results indicated differential expression of auxin-related TF genes between the barley genotypes in both the explants and the derived cultures. In support of the role of auxin in barley regeneration, distinct differences in the accumulation of free and oxidized auxin were observed in explants and explant-derived callus cultures of barley genotypes. Following the assumption that modifying gene expression might improve plant regeneration in barley, we treated the barley explants with trichostatin A (TSA), which affects histone acetylation. The effects of TSA were genotype-dependent as TSA treatment improved plant regeneration in two barley cultivars. TSA-induced changes in plant regeneration were associated with the increased expression of auxin biosynthesis-involved TFs. The study demonstrated that explant treatment with chromatin modifiers such as TSA might provide a new and effective epigenetic approach to improving plant regeneration in recalcitrant barley genotypes.

Aluminum or Low pH - Which Is the Bigger Enemy of Barley? Transcriptome Analysis of Barley Root Meristem Under Al and Low pH Stress.

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.

Albino Plant Formation in Androgenic Cultures: An Old Problem and New Facts.

High frequency of albino plant formation in isolated microspore or anther cultures is a great problem limiting the possibility of their exploitation on a wider scale. It is highly inconvenient as androgenesis-based doubled haploid (DH) technology provides the simplest and shortest way to total homozygosity, highly valued by plant geneticists, biotechnologists and especially, plant breeders, and this phenomenon constitutes a serious limitation of these otherwise powerful tools. The genotype-dependent tendency toward albino plant formation is typical for many monocotyledonous plants, including cereals like wheat, barley, rice, triticale, oat and rye - the most important from the economical point of view. Despite many efforts, the precise mechanism underlying chlorophyll deficiency has not yet been elucidated. In this chapter, we review the data concerning molecular and physiological control over proper/disturbed chloroplast biogenesis, old hypotheses explaining the mechanism of chlorophyll deficiency, and recent studies which shed new light on this phenomenon.

Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture.

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 tRNAGlu, 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.

Methyl Jasmonate Affects Photosynthesis Efficiency, Expression of HvTIP Genes and Nitrogen Homeostasis in Barley.

Jasmonates modulate many growth and developmental processes and act as stress hormones that play an important role in plant tolerance to biotic and abiotic stresses. Therefore, there is a need to identify the genes that are regulated through the jasmonate signalling pathway. Aquaporins, and among them the Tonoplast Intrinsic Proteins (TIPs), form the channels in cell membranes that are responsible for the precise regulation of the movement of water and other substrates between cell compartments. We identified the cis-regulatory motifs for the methyl jasmonate (MeJA)-induced genes in the promoter regions of all the HvTIP genes, which are active in barley seedlings, and thus we hypothesised that the HvTIP expression could be a response to jasmonate signalling. In the presented study, we determined the effect of methyl jasmonate on the growth parameters and photosynthesis efficiency of barley seedlings that had been exposed to different doses of MeJA (15-1000 µM × 120 h) in a hydroponic solution. All of the applied MeJA concentrations caused a significant reduction of barley seedling growth, which was most evident in the length of the first leaf sheath and dry leaf weight. The observed decrease of the PSII parameters after the exposure to high doses of MeJA (500 µM or higher) was associated with the downregulation of HvPsbR gene encoding one of the extrinsic proteins of the Oxygen Evolving Complex. The reduced expression of HvPsbR might lead to the impairment of the OEC action, manifested by the occurrence of the K-band in an analysis of fluorescence kinetics after MeJA treatment as well as reduced photosynthesis efficiency. Furthermore, methyl jasmonate treatment caused a decrease in the nitrogen content in barley leaves, which was associated with an increased expression the four tonoplast aquaporin genes (HvTIP1;2, HvTIP2;2, HvTIP4;1 and HvTIP4;2) predicted to transport the nitrogen compounds from the vacuole to the cytosol. The upregulation of the nitrogen-transporting HvTIPs might suggest their involvement in the vacuolar unloading of ammonia and urea, which both could be remobilised when the nitrogen content in the leaves decreases. Our research provides tips on physiological role of the individual TIP subfamily members of aquaporins under methyl jasmonate action.

Plastid differentiation during microgametogenesis determines green plant regeneration in barley microspore culture.

Developing plants from in vitro culture of microspores or immature pollen grains (androgenesis) is a highly genotype-dependent process whose effectiveness in cereals is significantly reduced by occurrence of albino regenerants. Here, we examined a hypothesis that the molecular differentiation of plastids in barley microspores prior to in vitro culture affects the genotype ability to regenerate green plants in culture. At the mid-to-late uninucleate (ML) stage, routinely used to initiate microspore culture, the expression of most genes involved in plastid transcription, translation and starch synthesis was significantly higher in microspores of barley cv. 'Mercada' producing 90% albino regenerants, than in cv. 'Jersey' that developed 90% green regenerants. The ML microspores of cv. 'Mercada' contained a large proportion of amyloplasts filled with starch, while in cv. 'Jersey' there were only proplastids. Using additional spring barley genotypes that differed in their ability to regenerate green plants we confirmed the correlation between plastid differentiation prior to culture and albino regeneration in culture. The expression of GBSSI gene (Granule-bound starch synthaseI) in early-mid (EM) microspores was a good marker of a genotype potential to produce green regenerants during androgenesis. Initiating culture from EM microspores that significantly improved regeneration of green plants may overcome the problem of albinism.

HorTILLUS-A Rich and Renewable Source of Induced Mutations for Forward/Reverse Genetics and Pre-breeding Programs in Barley (Hordeum vulgare L.).

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.

Mutation in HvCBP20 (Cap Binding Protein 20) Adapts Barley to Drought Stress at Phenotypic and Transcriptomic Levels.

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.