14 research outputs found
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
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 e ect of methyl jasmonate on the growth parameters and photosynthesis
e ciency of barley seedlings that had been exposed to di erent doses of MeJA (15–1000 M 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 M 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 e ciency. 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
Mutation in HvCBP20 (Cap binding protein 20) adapts barley to drought stress at phenotypic and transcriptomic levels
This work was supported by the European Regional Development Fund through the Innovative Economy for Poland 2007–2013, project WND-POIG.01.03.01-00-101/08 POLAPGEN-BD “Biotechnological tools for breeding cereals with increased resistance to drought,” task 22; National Science Centre, Poland, project SONATA 2015/19/D/NZ9/03573 “Translational genomics approach to identify the mechanisms of CBP20 signalosome in Arabidopsis and barley under drought stress.”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.European Regional Development Fund; National Science Centre, Polan
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 RNAseq
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 mM of bioavailable Al3C 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, Al3C 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
Use of molecular biology techniques to identify genetically modified organisms (GMOs) in food products – do you know what you eat?
tekst w j. pol. i ang.Ocena poprawności oznaczeń produktów spożywczych pod kątem zawartości genetycznie modyfikowanych organizmów (GMO)
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 182.2Mb 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 platformis 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
Table2.DOCX
<p>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 (NaN<sub>3</sub>) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M<sub>2</sub> plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M<sub>3</sub> progeny of 3,481 M<sub>2</sub> 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 M<sub>2</sub> 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.</p
Image3.tif
<p>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 (NaN<sub>3</sub>) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M<sub>2</sub> plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M<sub>3</sub> progeny of 3,481 M<sub>2</sub> 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 M<sub>2</sub> 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.</p