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1.
Plant Cell Environ ; 2024 Jul 15.
Article in English | MEDLINE | ID: mdl-39007549

ABSTRACT

Aluminum-dependent stoppage of root growth requires the DNA damage response (DDR) pathway including the p53-like transcription factor SUPPRESSOR OF GAMMA RADIATION 1 (SOG1), which promotes terminal differentiation of the root tip in response to Al dependent cell death. Transcriptomic analyses identified Al-induced SOG1-regulated targets as candidate mediators of this growth arrest. Analysis of these factors either as loss-of-function mutants or by overexpression in the als3-1 background shows ERF115, which is a key transcription factor that in other scenarios is rate-limiting for damaged stem cell replenishment, instead participates in transition from an actively growing root to one that has terminally differentiated in response to Al toxicity. This is supported by a loss-of-function erf115 mutant raising the threshold of Al required to promote terminal differentiation of Al hypersensitive als3-1. Consistent with its key role in stoppage of root growth, a putative ERF115 barley ortholog is also upregulated following Al exposure, suggesting a conserved role for this ATR-dependent pathway in Al response. In contrast to other DNA damage agents, these results show that ERF115 and likely related family members are important determinants of terminal differentiation of the root tip following Al exposure and central outputs of the SOG1-mediated pathway in Al response.

2.
Plant Signal Behav ; 19(1): 2371693, 2024 Dec 31.
Article in English | MEDLINE | ID: mdl-38923879

ABSTRACT

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.


Subject(s)
Droughts , Hordeum , Mutation , Plant Growth Regulators , Hordeum/genetics , Hordeum/metabolism , Hordeum/growth & development , Plant Growth Regulators/metabolism , Mutation/genetics , Gibberellins/metabolism , Gene Expression Regulation, Plant , Brassinosteroids/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Stress, Physiological/genetics , Lactones/metabolism
3.
Front Plant Sci ; 14: 1160695, 2023.
Article in English | MEDLINE | ID: mdl-37674734

ABSTRACT

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.

4.
Int J Mol Sci ; 24(3)2023 Jan 26.
Article in English | MEDLINE | ID: mdl-36768727

ABSTRACT

DNA damage is induced by many factors, some of which naturally occur in the environment. Because of their sessile nature, plants are especially exposed to unfavorable conditions causing DNA damage. In response to this damage, the DDR (DNA damage response) pathway is activated. This pathway is highly conserved between eukaryotes; however, there are some plant-specific DDR elements, such as SOG1-a transcription factor that is a central DDR regulator in plants. In general, DDR signaling activates transcriptional and epigenetic regulators that orchestrate the cell cycle arrest and DNA repair mechanisms upon DNA damage. The cell cycle halts to give the cell time to repair damaged DNA before replication. If the repair is successful, the cell cycle is reactivated. However, if the DNA repair mechanisms fail and DNA lesions accumulate, the cell enters the apoptotic pathway. Thereby the proper maintenance of DDR is crucial for plants to survive. It is particularly important for agronomically important species because exposure to environmental stresses causing DNA damage leads to growth inhibition and yield reduction. Thereby, gaining knowledge regarding the DDR pathway in crops may have a huge agronomic impact-it may be useful in breeding new cultivars more tolerant to such stresses. In this review, we characterize different genotoxic agents and their mode of action, describe DDR activation and signaling and summarize DNA repair mechanisms in plants.


Subject(s)
DNA Damage , Plant Breeding , DNA Repair , Plants/genetics , Plants/metabolism , Transcription Factors/metabolism
5.
BMC Genomics ; 23(1): 177, 2022 Mar 04.
Article in English | MEDLINE | ID: mdl-35246029

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.


Subject(s)
Hordeum , Gene Expression Regulation, Plant , Genotype , Heat-Shock Response/genetics , High-Throughput Nucleotide Sequencing , Hordeum/genetics , Phenotype
6.
Int J Mol Sci ; 22(24)2021 Dec 14.
Article in English | MEDLINE | ID: mdl-34948205

ABSTRACT

Root hairs play a crucial role in anchoring plants in soil, interaction with microorganisms and nutrient uptake from the rhizosphere. In contrast to Arabidopsis, there is a limited knowledge of root hair morphogenesis in monocots, including barley (Hordeum vulgare L.). We have isolated barley mutant rhp1.e with an abnormal root hair phenotype after chemical mutagenesis of spring cultivar 'Sebastian'. The development of root hairs was initiated in the mutant but inhibited at the very early stage of tip growth. The length of root hairs reached only 3% of the length of parent cultivar. Using a whole exome sequencing (WES) approach, we identified G1674A mutation in the HORVU1Hr1G077230 gene, located on chromosome 1HL and encoding a cellulose synthase-like C1 protein (HvCSLC1) that might be involved in the xyloglucan (XyG) synthesis in root hairs. The identified mutation led to the retention of the second intron and premature termination of the HvCSLC1 protein. The mutation co-segregated with the abnormal root hair phenotype in the F2 progeny of rhp1.e mutant and its wild-type parent. Additionally, different substitutions in HORVU1Hr1G077230 were found in four other allelic mutants with the same root hair phenotype. Here, we discuss the putative role of HvCSLC1 protein in root hair tube elongation in barley.


Subject(s)
Hordeum/genetics , Plant Roots/genetics , Alleles , Gene Expression Regulation, Plant/genetics , Mutation/genetics , Phenotype , Plant Proteins/genetics , Rhizosphere , Exome Sequencing/methods
7.
Cells ; 10(8)2021 08 05.
Article in English | MEDLINE | ID: mdl-34440762

ABSTRACT

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.


Subject(s)
Abscisic Acid/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Basic-Leucine Zipper Transcription Factors/metabolism , Plant Development , Plants, Genetically Modified/metabolism , Stress, Physiological , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Basic-Leucine Zipper Transcription Factors/genetics , Flowers/genetics , Flowers/growth & development , Flowers/metabolism , Fruit/genetics , Fruit/growth & development , Fruit/metabolism , Gene Expression Regulation, Plant , Germination , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , Seeds/genetics , Seeds/growth & development , Seeds/metabolism , Signal Transduction
8.
Front Genet ; 12: 675260, 2021.
Article in English | MEDLINE | ID: mdl-34220949

ABSTRACT

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.

9.
Methods Mol Biol ; 2288: 3-23, 2021.
Article in English | MEDLINE | ID: mdl-34270002

ABSTRACT

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.


Subject(s)
Edible Grain/growth & development , Edible Grain/physiology , Pigmentation , Plant Breeding/methods , Chlorophyll/deficiency , Chlorophyll/genetics , Diploidy , Edible Grain/genetics , Haploidy , Homozygote , Models, Biological , Molecular Biology/methods , Pigmentation/genetics , Pigments, Biological/deficiency , Pigments, Biological/genetics , Pollen/genetics , Pollen/growth & development , Pollen/physiology , Regeneration/genetics , Regeneration/physiology
10.
BMC Plant Biol ; 21(1): 22, 2021 Jan 07.
Article in English | MEDLINE | ID: mdl-33413097

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 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.


Subject(s)
Cell Differentiation/genetics , Chloroplasts/genetics , Color , Hordeum/growth & development , Hordeum/genetics , Organelle Biogenesis , Pollen/growth & development , Pollen/genetics , Cell Culture Techniques , Chloroplasts/physiology , Crops, Agricultural/genetics , Crops, Agricultural/growth & development , Genetic Variation , Genotype
11.
Int J Mol Sci ; 21(22)2020 Nov 12.
Article in English | MEDLINE | ID: mdl-33198069

ABSTRACT

ATR, a DNA damage signaling kinase, is required for cell cycle checkpoint regulation and detecting DNA damage caused by genotoxic factors including Al3+ ions. We analyzed the function of the HvATR gene in response to chemical clastogen-maleic acid hydrazide (MH). For this purpose, the Al-tolerant barley TILLING mutant hvatr.g was used. We described the effects of MH on the nuclear genome of hvatr.g mutant and its WT parent cv. "Sebastian", showing that the genotoxic effect measured by TUNEL test and frequency of cells with micronuclei was much stronger in hvatr.g than in WT. MH caused a significant decrease in the mitotic activity of root cells in both genotypes, however this effect was significantly stronger in "Sebastian". The impact of MH on the roots cell cycle, analyzed using flow cytometry, showed no differences between the mutant and WT.


Subject(s)
Aluminum/pharmacology , Ataxia Telangiectasia Mutated Proteins/metabolism , DNA Damage/drug effects , Hordeum/drug effects , Maleic Hydrazide/pharmacology , Arabidopsis Proteins/metabolism , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Nucleus/drug effects , Cell Nucleus/genetics , DNA Damage/genetics , Genome, Plant/drug effects , Genome, Plant/genetics , Genotype , Hordeum/genetics , Micronuclei, Chromosome-Defective/drug effects , Mutagens/pharmacology , Mutation/drug effects , Mutation/genetics , Plant Roots/drug effects , Plant Roots/genetics
12.
Plant Sci ; 300: 110593, 2020 Nov.
Article in English | MEDLINE | ID: mdl-33180718

ABSTRACT

CBP20 (Cap-Binding Protein 20) encodes a small subunit of nuclear Cap-Binding Complex (nCBC) that together with CBP80 binds mRNA cap. We previously described barley hvcbp20.ab mutant that demonstrated higher leaf water content and faster stomatal closure than the WT after drought stress. Hence, we presumed that the better water-saving mechanism in hvcbp20.ab may result from the lower permeability of epidermis that together with stomata action limit the water evaporation under drought stress. We asked whether hvcbp20.ab exhibited any differences in wax load on the leaf surface when subjected to drought in comparison to WT cv. 'Sebastian'. To address this question, we investigated epicuticular wax structure and chemical composition under drought stress in hvcbp20.ab mutant and its WT. We showed that hvcbp20.ab mutant exhibited the increased deposition of cuticular wax. Moreover, our gene expression results suggested a role of HvCBP20 as a negative regulator of both, the biosynthesis of waxes at the level of alkane-forming, and waxes transportation. Interestingly, we also observed increased wax deposition in Arabidopsis cbp20 mutant exposed to drought, which allowed us to describe the CBP20-regulated epicuticular wax accumulation under drought stress in a wider evolutionarily context.


Subject(s)
Dehydration/physiopathology , Hordeum/genetics , Hordeum/metabolism , Plant Epidermis/metabolism , Plant Proteins/metabolism , RNA Cap-Binding Proteins/metabolism , Water/metabolism , Dehydration/genetics , Gene Expression Regulation, Plant , Mutation , Plant Epidermis/genetics , Plant Proteins/genetics , RNA-Binding Proteins/metabolism , Stress, Physiological/genetics , Transcription Factors/metabolism
13.
Front Plant Sci ; 11: 1138, 2020.
Article in English | MEDLINE | ID: mdl-32849699

ABSTRACT

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.

14.
Int J Mol Sci ; 21(12)2020 Jun 18.
Article in English | MEDLINE | ID: mdl-32570736

ABSTRACT

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.


Subject(s)
Acetates/pharmacology , Cyclopentanes/pharmacology , Hordeum/physiology , Membrane Proteins/genetics , Oxylipins/pharmacology , Plant Growth Regulators/pharmacology , Plant Proteins/genetics , Gene Expression Profiling , Gene Expression Regulation, Plant/drug effects , Hordeum/drug effects , Membrane Proteins/metabolism , Nitrogen/metabolism , Photosynthesis/drug effects , Plant Proteins/metabolism , Promoter Regions, Genetic , Seeds/drug effects , Seeds/physiology
15.
Plant Cell Environ ; 43(9): 2239-2253, 2020 09.
Article in English | MEDLINE | ID: mdl-32501539

ABSTRACT

Strigolactones (SLs) are a group of plant hormones involved in many aspects of plant development and stress adaptation. Here, we investigated the drought response of a barley (Hordeum vulgare L.) mutant carrying a missense mutation in the gene encoding the SL-specific receptor HvD14. Our results clearly showed that hvd14.d mutant is hyper-sensitive to drought stress. This was illustrated by a lower leaf relative water content (RWC), impaired photosynthesis, disorganization of chloroplast structure, altered stomatal density and slower closure of stomata in response to drought in the mutant compared to the wild type parent cultivar Sebastian. Although the content of abscisic acid (ABA) and its derivatives remained unchanged in the mutant, significant differences in expression of genes related to ABA biosynthesis were observed. Moreover, hvd14.d was insensitive to ABA during seed germination. Analysis of Arabidopsis thaliana mutant atd14-1 also demonstrated that mutation in the SL receptor resulted in increased sensitivity to drought. Our results indicate that the drought-sensitive phenotype of barley SL mutant might be caused by a disturbed ABA metabolism and/or signalling pathways. These results together uncovered a link between SL signalling and ABA-dependent drought stress response in barley.


Subject(s)
Abscisic Acid/metabolism , Arabidopsis/physiology , Heterocyclic Compounds, 3-Ring/metabolism , Hordeum/physiology , Lactones/metabolism , Plant Proteins/genetics , Abscisic Acid/pharmacology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Dehydration , Droughts , Gene Expression Regulation, Plant , Germination/drug effects , Germination/physiology , Hordeum/drug effects , Mutation , Photosystem II Protein Complex/metabolism , Plant Proteins/metabolism , Plant Stomata/physiology , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Seeds/drug effects , Seeds/physiology , Signal Transduction/genetics
16.
Plant Sci ; 291: 110321, 2020 Feb.
Article in English | MEDLINE | ID: mdl-31928659

ABSTRACT

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.


Subject(s)
Gametogenesis, Plant/physiology , Hordeum/physiology , Plastids/physiology , Pollen , Regeneration , Tissue Culture Techniques
17.
PLoS One ; 14(12): e0226423, 2019.
Article in English | MEDLINE | ID: mdl-31846477

ABSTRACT

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.


Subject(s)
Aquaporins/genetics , Droughts , Hordeum/genetics , Hordeum/physiology , Stress, Physiological/genetics , Water/pharmacology , Amino Acid Sequence , Aquaporins/chemistry , Gene Expression Regulation, Plant/drug effects , Hordeum/drug effects , Hordeum/metabolism , Plant Growth Regulators/metabolism , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , Stress, Physiological/drug effects
18.
Int J Mol Sci ; 20(24)2019 Dec 05.
Article in English | MEDLINE | ID: mdl-31817496

ABSTRACT

Root systems play a pivotal role in coupling with drought stress, which is accompanied with a substantial transcriptome rebuilding in the root tissues. Here, we present the results of global gene expression profiling of roots of two barley genotypes with contrasting abilities to cope with drought that were subjected to a mild level of the stress. We concentrate our analysis on gene expression regulation processes, which allowed the identification of 88 genes from 39 families involved in transcriptional regulation in roots upon mild drought. They include 13 genes encoding transcription factors (TFs) from AP2 family represented by ERFs, DREB, or B3 domain-containing TFs, eight WRKYs, six NACs, five of the HD-domain, MYB or MYB-related, bHLH and bZIP TFs. Also, the representatives of C3H, CPP, GRAS, LOB-domain, TCP, Tiffy, Tubby, and NF-Ys TFs, among others were found to be regulated by the mild drought in barley roots. We found that drought tolerance is accompanied with a lower number of gene expression changes than the amount observed in a susceptible genotype. The better drought acclimation may be related to the activation of transcription factors involved in the maintenance of primary root growth and in the epigenetic control of chromatin and DNA methylation. In addition, our analysis pointed to fives TFs from ERF, LOB, NAC, WRKY and bHLH families that may be important in the mild but not the severe drought response of barley roots.


Subject(s)
Gene Expression Regulation, Plant , Hordeum , Plant Proteins , Plant Roots , Stress, Physiological , Transcriptome , Dehydration/genetics , Dehydration/metabolism , Gene Expression Profiling , Hordeum/genetics , Hordeum/metabolism , Plant Proteins/biosynthesis , Plant Proteins/genetics , Plant Roots/genetics , Plant Roots/metabolism
19.
Front Plant Sci ; 10: 1299, 2019.
Article in English | MEDLINE | ID: mdl-31695712

ABSTRACT

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.

20.
G3 (Bethesda) ; 9(8): 2657-2666, 2019 08 08.
Article in English | MEDLINE | ID: mdl-31213514

ABSTRACT

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.


Subject(s)
Genomics , High-Throughput Nucleotide Sequencing , Coffea/genetics , Genomics/methods , High-Throughput Nucleotide Sequencing/methods , Mutation , Polymerase Chain Reaction , Polymorphism, Single Nucleotide
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